Enzymatic synthesis of poly(amine-co-esters) and methods of use thereof for gene delivery

ABSTRACT

Poly(amine-co-ester) polymers, methods of forming active agent-load nanoparticles therefrom, and methods of using the nanoparticles for drug delivery are disclosed. The nanoparticles can be coated with an agent that reduces surface charge, an agent that increases cell-specific targeting, or a combination thereof. Typically, the loaded nanoparticles are less toxic, more efficient at drug delivery, or a combination thereof compared to a control other transfection reagents. In some embodiments, the nanoparticles are suitable for in vivo delivery, and can be administered systemically to a subject to treat a disease or condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/293,733, filedJun. 2, 2014, now U.S. Pat. No. 9,272,043, which claims priority toInternational Patent Application No. PCT/US2012/067447 entitled“Enzymatic Synthesis of Novel poly(amine-co-esters) and Methods of useThereof for Gene Delivery”, filed Nov. 30, 2012, which claims priorityto U.S. Provisional Application No. 61/566,412 entitled “EnzymaticSynthesis of Novel poly(amine-co-esters) and Their Use as HighlyEfficient Non-viral Vectors for Gene Delivery” filed Dec. 2, 2011. Thisapplication also claims priority to U.S. Provisional Application No.61/870,497 entitled “Enzymatic Synthesis of Novel poly(amine-co-esters)and Methods of use Thereof for Gene Delivery” filed Aug. 27, 2013. Wherepermissible, these applications are incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Agreement R56EB000487 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on Jan. 5, 2016 as a text file named“YU_5543_ST25.txt,” created on Aug. 5, 2014, and having a size of 2,819bytes is hereby incorporated by reference pursuant to 37 C.F.R.§1.52(e)(5).

FIELD OF THE INVENTION

The field of the invention is generally related to novel polymercompositions and methods for improved systemic delivery of nucleic acidsin vivo.

BACKGROUND OF THE INVENTION

Non-viral vectors for gene delivery have attracted much attention in thepast several decades due to their potential for limited immunogenicity,ability to accommodate and deliver large size genetic materials, andpotential for modification of their surface structures. Major categoriesof non-viral vectors include cationic lipids and cationic polymers.Cationic lipid-derived vectors, which were pioneered by Felgner andcolleagues, represent some of the most extensively investigated systemsfor non-viral gene delivery (Felgner, et al. Lipofection: a highlyefficient, lipid-mediated DNA-transfection procedure. PNAS, 84,7413-7417 (1987)) (Templeton, et al. Improved DNA: liposome complexesfor increased systemic delivery and gene expression. Nat. Biotechnol.15, 647-652 (1997)) (Chen, et al. Targeted nanoparticles deliver siRNAto melanoma. J. Invest. Dermatol. 130, 2790-2798 (2010)).

Cationic polymer non-viral vectors have gained increasing attentionbecause of flexibility in their synthesis and structural modificationsfor specific biomedical applications. Both cationic lipid and cationicpolymer systems deliver genes by forming condensed complexes withnegatively charged DNA through electrostatic interactions: complexformation protects DNA from degradation and facilitates its cellularuptake and intracellular traffic into the nucleus.

Polyplexes formed between cationic polymers and DNA are generally morestable than lipoplexes formed between cationic lipids and DNA, but bothare often unstable in physiological fluids, which contain serumcomponents and salts, and tend to cause the complexes to break apart oraggregate (Al-Dosari, et al. Nonviral gene delivery: principle,limitations, and recent progress. AAPS J. 11, 671-681 (2009)) (Tros deIlarduya, et al. Gene delivery by lipoplexes and polyplexes. Eur. J.Pharm. Sci. 40, 159-170 (2010)). Additionally, although some workindicates that anionic polymers or even naked DNA can provide some levelof transfection under certain conditions, transfection by both lipidsand polymers usually requires materials with excess charge, resulting inpolyplexes or lipoplexes with net positive charges on the surface(Nicol, et al. Poly-L-glutamate, an anionic polymer, enhances transgeneexpression for plasmids delivered by intramuscular injection with invivo electroporation. Gene. Ther. 9, 1351-1358 (2002)) (Schlegel, et al.Anionic polymers for decreased toxicity and enhanced in vivo delivery ofsiRNA complexed with cationic liposomes. J. Contr. Rel. 152, 393-401(2011)) (Liu, et al, Nonviral gene delivery: What we know and what isnext. AAPS J. 9, E92-E104 (2007)) (Liu, et al. Hydrodynamics-basedtransfection in animals by systemic administration of plasmid DNA. GeneTher. 6, 1258-1266 (1999)). When injected into the circulatory system invivo, the positive surface charge initiates rapid formation of complexaggregates with negatively charged serum molecules or membranes ofcellular components, which are then cleared by the reticuloendothelialsystem (RES).

More importantly, many cationic vectors developed so far exhibitsubstantial toxicity, which has limited their clinical applicability(Tros de Ilarduya, et al. Gene delivery by lipoplexes and polyplexes.Eur. J. Pharm. Sci. 40, 159-170 (2010)) (Gao, et al. The association ofautophagy with polyethylenimine-induced cytotoxity in nephritic andhepatic cell lines. Biomaterials 32, 8613-8625 (2011)) (Felgner, et al.Enhanced gene delivery and mechanism studies with a novel series ofcationic lipid formulations. J. Biol. Chem. 269, 2550-2561 (1994))(Kafil, et al. Cytotoxic Impacts of Linear and Branched PolyethylenimineNanostructures in A431 Cells. BioImpacts 1, 23-30 (2011)) (Lv, et al.Toxicity of cationic lipids and cationic polymers in gene delivery. JContr. Rel. 114, 100-109 (2006)). This too appears to depend on charge:excess positive charges on the surface of the complexes can interactwith cellular components, such as cell membranes, and inhibit normalcellular processes, such as clathrin-mediated endocytosis, activity ofion channels, membrane receptors, and enzymes or cell survival signaling(Gao, et al. The association of autophagy with polyethylenimine-inducedcytotoxity in nephritic and hepatic cell lines. Biomaterials 32,8613-8625 (2011)) (Felgner, et al. Enhanced gene delivery and mechanismstudies with a novel series of cationic lipid formulations. J. Biol.Chem. 269, 2550-2561 (1994)) (Kafil, et al. Cytotoxic Impacts of Linearand Branched Polyethylenimine Nanostructures in A431 Cells. BioImpacts1, 23-30 (2011)).

As a result, cationic lipids often cause acute inflammatory responses inanimals and humans, whereas cationic polymers, such as PEI, destabilizethe plasma-membrane of red blood cells and induce cell necrosis,apoptosis and autophagy (Tros de Ilarduya, et al. Gene delivery bylipoplexes and polyplexes. Eur. J. Pharm. Sci. 40, 159-170 (2010)) (Gao,et al. The association of autophagy with polyethylenimine-inducedcytotoxity in nephritic and hepatic cell lines. Biomaterials 32,8613-8625 (2011)) (Lv, et al. Toxicity of cationic lipids and cationicpolymers in gene delivery. J. Contr. Rel. 114, 100-109 (2006)). Becauseof these undesirable effects, there is a need for highly efficientnon-viral vectors that have lower charge densities.

Synthesis of a family of biodegradable poly(amine-co-esters) formed viaenzymatic copolymerization of diesters with amino-substituted diols isdiscussed in Liu, et al. Enzyme-synthesized poly(amine-co-esters) asnonviral vectors for gene delivery. J. Biomed. Mater. Res. A 96A,456-465 (2011) and Jiang, Z. Lipase-catalyzed synthesis ofpoly(amine-co-esters) via copolymerization of diester withamino-substituted diol. Biomacromolecules 11, 1089-1093 (2010).

Diesters with various chain length (e.g., from succinate tododecanedioate) were copolymerized with diethanolamines with either analkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituenton the nitrogen. The high tolerance of the lipase catalyst allowed thecopolymerization reactions to complete in one step without protectionand deprotection of the amino functional groups. Upon protonation atslightly acidic conditions, these poly(amine-co-esters) readily condenseDNA and form nano-sized polyplexes. Screening studies revealed that oneof these materials, poly(N-methyldiethyleneamine sebacate) (PMSC),transfected a variety of cells including HEK293, U87-MG, and 9 L, withefficiency comparable to that of leading commercial products, such asLipofectamine 2000 and PEI14. PMSC had been previously used for genedelivery, but the delivery efficiency of the enzymatically synthesizedmaterials was approximately five orders of magnitude higher than anypreviously reported (Wang, et al. Synthesis and characterization ofcationic micelles self-assembled from a biodegradable copolymer for genedelivery. Biomacromolecules 8, 1028-1037 (2007)) (Wang, et al. Theself-assembly of biodegradable cationic polymer micelles as vectors forgene transfection. Biomaterials 28, 5358-5368 (2007)). However, thesepoly(amine-co-esters) were not effective for systemic delivery ofnucleic acids in vivo. This may be due to the fact that the polyplexesformed by these polymers and genetic materials (1) do not havesufficient efficiency for in vivo applications and/or (2) are not stableenough in the blood and fall apart or aggregate during circulation.

Accordingly, there remains a need for non-viral vectors suitable forefficient systemic, in vivo delivery of nucleic acids with low toxicity.

There is also a need for polymeric nanocarriers which can be prepared inas few steps as possible and in which the molecular weight and/orpolymer composition can be easily controlled.

Therefore, it is an object of the invention to provide improved polymerswhich can effectively deliver therapeutic, diagnostic, and/orprophylactic agents in vivo, and methods of making and using thereof.

It is an object of the invention to provide improved polymers which caneffectively deliver genetic materials to cells in high efficiency invitro and are suitable for in vivo delivery of nucleic acids, andmethods of making thereof.

It is also an object of the invention to provide methods of usingimproved polymers for systemic delivery of nucleic acids in vivo.

SUMMARY OF THE INVENTION

Polymers with improved properties for delivering therapeutic,diagnostic, and/or prophylactic agents are described.

Polymers having the formula below are disclosed.

wherein each occurrence of n is an integer from 1-30, each occurrence ofm, o, and p is independently an integer from 1-20, and each occurrenceof x, y, and q is independently an integer from 1-1000, and Z is O orNR′, wherein R′ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted aryl.

In some embodiments, Z is O.

In some embodiments, Z is O and n is an integer from 1-16, such as 4,10, 13, or 14.

In some embodiments, Z is O, n is an integer from 1-16, such as 4, 10,13, or 14, and m is an integer from 1-10, such as 4, 5, 6, 7, or 8.

In some embodiments, Z is O, n is an integer from 1-16, such as 4, 10,13, or 14, m is an integer from 1-10, such as 4, 5, 6, 7, or 8, and oand p are the same integer from 1-6, such 2, 3, or 4.

In some embodiments, Z is O, n is an integer from 1-16, such as 4, 10,13, or 14, m is an integer from 1-10, such as 4, 5, 6, 7, or 8, and R isalkyl, such a methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl,or aryl, such as phenyl.

In certain embodiments, n is 14 (e.g., pentadecalactone, PDL), m is 7(e.g., diethylsebacate, DES), o and p are 2 (e.g.,N-methyldiethanolamine, MDEA). In certain embodiments, n, m, o, and pare as defined above, and PEG is incorporated as a monomer.

The polymer is preferably biocompatible. Readily available lactones ofvarious ring sizes are known to possess low toxicity: for example,polyesters prepared from small lactones, such as poly(caprolactone) andpoly(p-dioxanone) are commercially available biomaterials which havebeen used in clinical applications. Large (e.g., C₁₆-C₂₄) lactones andtheir polyester derivatives are natural products that have beenidentified in living organisms, such as bees.

The polymers can further include a block of an alkylene oxide, such aspolyethylene oxide, polypropylene oxide, and/or polyethyleneoxide-co-polypropylene oxide. The structure of a PEG-containing diblockpolymer is shown below:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, x, y, q, and w are independently integers from1-1000, and Z is O or NR′, wherein R′ is hydrogen, substituted orunsubstituted alkyl, or substituted or unsubstituted aryl. In particularembodiments, the values of x, y, q, and w are such that the weightaverage molecular weight of the polymer is greater than 20,000 Daltons.

The structure of a PEG-containing triblock copolymer is shown below:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, x, y, q, and w are independently integers from1-1000, and Z is O or NR′, wherein R′ is hydrogen, substituted orunsubstituted alkyl, or substituted or unsubstituted aryl. In particularembodiments, the values of x, y, q, and w are such that the weightaverage molecular weight of the polymer is greater than 20,000 Daltons.

The blocks of polyalkylene oxide can located at the termini of thepolymer (i.e., by reacting PEG having one hydroxy group blocked, forexample, with a methoxy group), within the polymer backbone (i.e.,neither of the hydroxyl groups are blocked), or combinations thereof.

In particular embodiments, the values of x, y, q, and/or w are such thatthe weight average molecular weight of the polymer is greater than20,000 Daltons.

The polymer can prepared from one or more lactones, one or moreamine-diols (Z═O) or triamines (Z═NR′), and one or more diacids ordiesters. In those embodiments where two or more different lactone,diacid or diester, and/or triamine or amine-diol monomers are used, thanthe values of n, o, p, and/or m can be the same or different.

The monomers show above can be unsubstituted or can be substituted.“Substituted”, as used herein, means one or more atoms or groups ofatoms on the monomer has been replaced with one or more atoms or groupsof atoms which are different than the atom or group of atoms beingreplaced. In some embodiments, the one or more hydrogens on the monomeris replaced with one or more atoms or groups of atoms. Examples offunctional groups which can replace hydrogen are listed above in thedefinition. In some embodiments, one or more functional groups can beadded which vary the chemical and/or physical property of the resultingmonomer/polymer, such as charge or hydrophilicity/hydrophobicity, etc.Exemplary substituents include, but are not limited to, halogen,hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or anacyl), thiocarbonyl (such as a thioester, a thioacetate, or athioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate,amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, nitro,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

The polymers can be used to form micro- and/or nanoparticles havingencapsulated therein therapeutic, diagnostic, and/or prophylactic agent.The agent to be encapsulated and delivered can be a small molecule agent(i.e., non-polymeric agent having a molecular weight less than 2,000,1500, 1,000, 750, or 500 Dalton) or a macromolecule (e.g., an oligomeror polymer) such as proteins, enzymes, peptides, nucleic acids, etc. Theparticles can be used for in vivo and/or in vitro delivery of the agent.

The particles prepared from the polymers can be coated with surfacecharge altering materials, such as polypeptides, that increase stabilityand half-life of the particles in systemic circulation. The chargealtering material can include a targeting moiety that increasestargeting of the particles to a cell type or cell state of interest.

In some embodiments, the particles have a mean particle size from about100 nm to about 300 nm, preferably from about 150 nm to about 275 nm. Insome embodiments, the weight:weight ratio of polymer:polynucleotide isbetween about 25:1 and 250:1.

In some embodiments, the polymers can be used to form polymericnanoparticulate polynucleotide carriers, referred to herein aspolyplexes, which are effective for delivering the polynucleotides tocells in vitro and in vivo. The polyplexes have improved efficacy orreduced toxicity in vivo compared to other polynucleotide deliveryapproaches, enabling the polyplexes to be utilized in a broad range oftherapeutic applications, for example, gene therapy. Typically, thepolyplexes are less toxic and more efficient at transfectingpolynucleotides when compared to a control, such as LIPOFECTAMINE 2000or polyethylenimine (PEI). In some embodiments, the polyplexes aresuitable for in vivo transfection, and can be used when othertransfection reagents are too toxic or too inefficient to support invivo applications. In some embodiments, the in vivo application includessystemic administration of the polyplexes.

The polyplexes can be coated with one or more agents that reduce thesurface charge of the polyplex at physiological pH. The coating canimpart a neutral or negative surface charge to the polyplex. The agentcan include, for example, a polypeptide with a series of negativelycharged amino acids, such as glutamic acids or aspartic acids. In someembodiments, the polypeptide includes a cell targeting signal or celltargeting domain that enhances targeting of the polyplexes to a specificcell-type or cell-state. For example, the cell targeting domain canenhance targeting of the polyplexes to cancer cells. Exemplary celltargeting domains include RGD, R/KxxR/K where “x” is any amino acid,GdPdLGdVdRG (SEQ ID NO:5), and ASGPR (SEQ ID NO:6). In some embodiments,the stretch of negatively charged amino acids and the cell targetingdomain are linked by a linker polypeptide. The linker can be a series ofglycines. An exemplary coating including an agent that reduces surfacecharge and provides cell specific targeting to cancer cells isEEEEEEEEEEEEEEEEGGGGGGRGDK (SEQ ID NO:1).

The polynucleotide can include a sequence that encodes a protein, asequence that encodes a functional nucleic acid, or can itself be afunctional nucleic acid, rRNA, or tRNA. Functional nucleic acidsinclude, but are not limited to, antisense molecules, siRNA, miRNA,aptamers, ribozymes, triplex forming molecules, RNAi, and external guidesequences. In some embodiments, the polynucleotide includes anexpression control sequence operably linked to a sequence encoding aprotein, functional nucleic acid, rRNA, or tRNA. For example, thepolynucleotide can be an expression vector.

Compositions, such as pharmaceutical compositions, containing theparticles are also disclosed. The particles can be contacted with cellsto transfect the agent, such as a polynucleotide, into the cells. Insome embodiments, the contacting occurs in vivo by administering theparticles, or a pharmaceutical composition containing the particles, toa subject in an effective amount to treat a disease or condition. Thedisease or condition can be, for example, a mitochondrial disease, aninfectious disease, a cancer, a metabolic disorder, an autoimmunedisease, an inflammatory disorder, or an age-related disorder. Theparticles can be administered parenterally, transdermally, ortransmucosally. The particles can be administered systemically orlocally.

In some embodiments, contacting the cells with polyplexes to transfectthe polynucleotide occurs in vitro, or ex vivo. The cells can be primarycells or cells from a cell line. The primary cells can be harvested froma subject. In some embodiments, the transfected cells are administeredback to the subject, or to a different subject as part of a cell-basedtherapy for treating a disease or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing a two-stage process for the preparation ofterpolymers from a lactone, DES, and MDEA.

FIGS. 2A and 2B are scatter plots of copolymer M_(w) over polymerizationtime (hours), and M_(w)/M_(n) over copolymer M_(w), respectively,illustrating variations of product molecular weight and polydispersityduring copolymerization of PDL with DES and MDEA at differenttemperatures (⋄=60° C., □=70° C., Δ=80° C., and X=90° C. Polymerizationconditions: 2:3:3 (molar ratio) PDL/DES/MDEA, 1.4 mmHg pressure.

FIG. 3A is a graph showing the gene deliver efficiency (RLU/mg protein)of various terpolymers (as labeled) on HEK293 cells (□) and A549 cells(⋄). FIG. 3B is a scatter plot showing the effect of III-20% PDL to DNAratio (ratio of polymer to DNA) on transfection efficiency on HEK293cells (RLU/mg protein). FIG. 3C is a line graph showing cell viability(percentage of control) as a function of PEI (□) or III-20% PDL(⋄)concentration (μg ml⁻¹) on HEK293 cells. FIG. 3D is a line graph showingcell viability (percentage of control) as a function of PEI (□) orIII-20% PDL(⋄) concentration (μg ml⁻¹) on A549 cells.

FIG. 4 is a graph showing the size of III-20% PDL/DNA complexes (radius)with different polymer-to-DNA ratios (Polymer:DNA ratio w/w).

FIG. 5 is a graph showing release of DNA (relative fluorescence unit(RFU) (%)) from III-20% PDL/DNA complexes (“terpolymer”) and PEI/DNAcomplexes (“PEI”) with (▪) and without (□) heparin. Heparin was added topolyplexes solution with a final concentration of 2% w/v. The values areexpressed as a percentage of the fluorescence obtained with naked DNA.Each value is the mean of duplicates.

FIG. 6 is a line graph showing RFU and absorbance of elution fractions(elution volume (ml)) following size exclusion chromatography todetermine the amount of III-20% PDL polymer associated with or withoutDNA when mixed with DNA at a 100:1 weight ratio. Elution fractions weremonitored for both III-20% PDL polymer (•) and Cy3-DNA (□) content afterloading III-20% PDL/Cy3-DNA polyplexes on a Sepharose CL-2B column.Representative data from three separate experiments are shown. Theamount of DNA-associated polymer was determined by area under curveanalysis.

FIG. 7 is a line graph showing RFU (%) as a function of polymer/DNA(w/w) in an ethidium bromide exclusion assay using III-20% PDL. Thevalues are expressed as a percentage of the fluorescence relative (RFU)to the initial fluorescence without polymer.

FIG. 8 is two line graphs showing (RFU (%)) of naked DNA (A) or III-20%PDL/DNA complexes (B) with (□) or without (▪) DNase degradation at 37°C. at various time points (incubation time (min). Residual DNA wasquantified using PICOGREEN (Invitrogen). The values are expressed as apercentage of the fluorescence obtained at time 0 min.

FIG. 9 is a histogram (top panel) and a bar graph (bottom panel) showingcell internalization of III-20% PDL/Cy3-DNA polyplexes as monitoredusing flow cytometry. For the bar graph, n=3, **p<0.005, *p<0.05 usingstudent's t-test with respect to polyplex with 10× coat.

FIG. 10 is a dot plot showing the toxicity of III-20% PDL/TRAIL,Lip2k/TRAIL and PEI/TRAIL complexes (growth inhibition %) on A549 tumorcells. Toxicity was determined five days after treatment by standard MTTassay.

FIGS. 11A and 11D are bar graphs showing the zeta potential (my) ofLip2k/DNA complexes (A) or PEI/DNA complexes (D) with no coat, or 2.5×,5×, or 10× coating of polyE-mRGD in the presence (shaded bars) orabsence (open bars) of 10% serum. FIGS. 11B and 11E are line graphsshowing the radius (nm) of Lip2k/DNA complexes (B) or PEI/DNA complexes(E) with no coat (▪), or 2.5× (•), 5× (▴), or 10× (▾) coating ofpolyE-mRGD. FIGS. 11C and 11F are dot plots showing the gene deliveryefficiency (RLU/mg protein) for Lip2k/DNA complexes (C) or PEI/DNAcomplexes (F) with no coat, or 2.5×, 5×, or 10× coating of polyE-mRGD.

FIG. 12 is a line graph showing the toxicity of naked DNA (▴) andpolyE-mRGD (▪) on A549 cells at the indicated concentrations. Toxicitywas determined five days after treatment by standard MTT assay.

FIG. 13A is a scatter plot comparing in vivo luciferase expression of aconstruct delivered by polyE-mRGD coated (with coat) and uncoated (nocoat) polyplexes transfection. Polyplexes were administrated throughtail vein injection and luciferase expression was determined 48 h afterthe last treatment of three consecutive daily treatments. FIG. 13B is abar graph showing the zeta potential (my) of polyplexes with no coat, or2.5×, 5×, or 10× coating of polyE-mRGD in the presence (shaded bars) orabsence (open bars) of 10% serum. FIG. 13C is a line graph showing thesize (radius (nm)) of polyplexes with no coat (□), or 2.5× (⋄), 5× (Δ),or 10× (∇) coating of polyE-mRGD determined by dynamic light scatteringat various time intervals. FIG. 13D is a dot plot showing the genedelivery efficiency (RLU/mg protein) for polyplexes with no coat, or2.5×, 5×, or 10× coating of polyE-mRGD.

FIG. 14A is a histogram showing the results of flow cytometry analysisof A549 cells 2 days after transfection with four pg of pEGFP plasmidDNA, using III-20% PDL, Lipofectamine 2000 and PEI. FIG. 14B is a linegraph showing weight (grams) of mice during a time course of tail veinadministration of PBS control (V), or III-20% PDL polyplexes with Lucwith polyE-mRGD coat (□), TRAIL no coat (◯), or TRAIL with polyE-mRGDcoat (Δ) three days a week, at a dose of 1.7 mg per mouse (based on themaximum dosage of polymer that can be used in 200 μl buffer), for 6weeks. FIG. 14C is a line graph showing tumor volume (mm³) of tumor onmice during a time course of tail vein administration of PBS control(∇), or III-20% PDL polyplexes with Luc with polyE-mRGD coat (□), TRAILno coat (⋄), or TRAIL with polyE-mRGD coat (Δ) three days a week, at adose of 1.7 mg per mouse (based on the maximum dosage of polymer thatcan be used in 200 μl buffer), for 6 weeks.

FIGS. 15A and 15B are thermogravimetric curves of the PEG2K-PPMS (A) andPEG5K-PPMS (B) copolymers with different PDL contents.

FIGS. 16A and 16B are DSC heating curves of (A) PEG2K-PPMS copolymerswith different PDL contents: (a) 11% PDL, (b) 20% PDL, (c) 30% PDL, (d)40% PDL, (e) 51% PDL; and (B) PEG5K-PPMS copolymers with different PDLcontents: (a) 10% PDL, (b) 20% PDL, (c) 30% PDL, (d) 41% PDL, (e) 50%PDL.

FIG. 17A is a TEM image (A) and FIG. 17B is a graph showing the particlesize distribution (B) of DTX-loaded PEG2K-PPMS-30% PDL copolymermicelles.

FIGS. 18A and 18B are graphs showing the variation in average size (FIG.18A) and zeta-potential (FIG. 18B) of blank PEG2K-PPMS copolymermicelles as a function of PBS medium pH.

FIGS. 19A and 19B are graphs showing variations of pyrene I3/I1intensity ratio as a function of logarithm of polymer concentration(mg/mL) for PEG2K-PPMS copolymer micelle samples in two different PBSsolutions (0.01 M): (FIG. 19A) PBS with pH of 7.4, (FIG. 19B) PBS withpH of 5.0.

FIGS. 20A and 20B are graphs showing the average micelle size vs.incubation time for PEG2K-PPMS copolymer micelles incubated in PBSsolution (0.01 M, pH=7.4) containing (FIG. 20A) 10 vol. % human serumsolution and (FIG. 20B) 10 vol. % FBS solution.

FIG. 21 is a graph showing the in vitro accumulative release of DTX fromthe drug-loaded PEG2K-PPMS copolymer micelles in PBS solution (0.01 M)with pH of 7.4 or 5.0.

FIG. 22 is a graph showing the relative uptake efficiency of free C6 andC6-loaded micelles of PEG2K-PPMS copolymers with various PDL contents bySK-BR-3 cells at C6 concentration of 0.2 μg/mL. The intracellular C6 MFIvalues were measured by flow cytometry after 1-8 h incubation and aregiven as mean±SD (n=3). *p<0.05 and **p<0.01 compared withPEG2K-PPMS-11% PDL.

FIGS. 23A-23C are graphs showing the viabilities of SK-BR-3 cells afterincubation for 48 h with various doses of PEG2K-PPMS copolymer micelles:(FIG. 23A) blank micelles (medium pH: 7.4), (FIG. 23B) DTX-loadedmicelles of the copolymers with different PDL contents (medium pH: 7.4),and (FIG. 23C) DTX-loaded micelles of the copolymer with 11% PDL (mediumpH: 7.4 or 6.5). Data are given as mean±SD (n=3). *p<0.05 and **p<0.01compared with PEG2K-PPMS-11% PDL copolymer sample.

FIG. 24 is a graph showing the hemolytic activity of blank PEG2K-PPMScopolymer micelles. Triton X-100 and PBS were used as the positive andnegative controls, respectively. Data are given as mean±SD (n=3).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “polyplex” as used herein refers to polymeric micro- and/ornanoparticles or micelles having encapsulated therein, dispersed within,and/or associated with the surface of, one or more polynucleotides.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

The terms “subject,” “individual,” and “patient” refer to any individualwho is the target of treatment using the disclosed compositions. Thesubject can be a vertebrate, for example, a mammal. Thus, the subjectcan be a human. The subjects can be symptomatic or asymptomatic. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, whether male or female, are intended to be covered. A subjectcan include a control subject or a test subject.

The term “biocompatible” as used herein refers to one or more materialsthat are neither themselves toxic to the host (e.g., an animal orhuman), nor degrade (if the material degrades) at a rate that producesmonomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host.

The term “biodegradable” as used herein means that the materialsdegrades or breaks down into its component subunits, or digestion, e.g.,by a biochemical process, of the material into smaller (e.g.,non-polymeric) subunits.

The term “diameter” is art-recognized and is used herein to refer toeither of the physical diameter or the hydrodynamic diameter. Thediameter of an essentially spherical particle may refer to the physicalor hydrodynamic diameter. The diameter of a nonspherical particle mayrefer preferentially to the hydrodynamic diameter. As used herein, thediameter of a non-spherical particle may refer to the largest lineardistance between two points on the surface of the particle. Whenreferring to multiple particles, the diameter of the particles typicallyrefers to the average diameter of the particles. Particle diameter canbe measured using a variety of techniques in the art including, but notlimited to, dynamic light scattering.

“Sustained release” as used herein refers to release of a substance overan extended period of time in contrast to a bolus type administration inwhich the entire amount of the substance is made biologically availableat one time.

The term “microspheres” is art-recognized, and includes substantiallyspherical colloidal structures, e.g., formed from biocompatible polymerssuch as subject compositions, having a size ranging from about one orgreater up to about 1000 microns. In general, “microcapsules,” also anart-recognized term, may be distinguished from microspheres, becausemicrocapsules are generally covered by a substance of some type, such asa polymeric formulation. The term “microparticles” is alsoart-recognized, and includes microspheres and microcapsules, as well asstructures that may not be readily placed into either of the above twocategories, all with dimensions on average of less than about 1000microns. A microparticle may be spherical or nonspherical and may haveany regular or irregular shape. If the structures are less than aboutone micron (1000 nm) in diameter, then the corresponding art-recognizedterms “nanosphere,” “nanocapsule,” and “nanoparticle” may be utilized.In certain embodiments, the nanospheres, nanocapsules and nanoparticleshave an average diameter of about 500 nm, 200 nm, 100 nm, 50 nm, 10 nm,or 1 nm. In some embodiments, the average diameter of the particles isfrom about 200 nm to about 600 nm, preferably from about 200 to about500 nm. Microparticles can be used for gene therapy, particularly forvaccinations.

A composition containing microparticles or nanoparticles may includeparticles of a range of particle sizes. In certain embodiments, theparticle size distribution may be uniform, e.g., within less than abouta 20% standard deviation of the mean volume diameter, and in otherembodiments, still more uniform, e.g., within about 10%, 8%, 5%, 3%, or2% of the median volume diameter.

The term “particle” as used herein refers to any particle formed of,having attached thereon or thereto, or incorporating a therapeutic,diagnostic or prophylactic agent, optionally including one or morepolymers, liposomes micelles, or other structural material. A particlemay be spherical or nonspherical. A particle may be used, for example,for diagnosing a disease or condition, treating a disease or condition,or preventing a disease or condition.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and includewithout limitation intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “surfactant” as used herein refers to an agent that lowers thesurface tension of a liquid.

The term “targeting moiety” as used herein refers to a moiety thatlocalizes to or away from a specific locale. The moiety may be, forexample, a protein, nucleic acid, nucleic acid analog, carbohydrate, orsmall molecule. Said entity may be, for example, a therapeutic compoundsuch as a small molecule, or a diagnostic entity such as a detectablelabel. Said locale may be a tissue, a particular cell type, or asubcellular compartment. In one embodiment, the targeting moiety directsthe localization of an active entity. The active entity may be a smallmolecule, protein, polymer, or metal. The active entity may be usefulfor therapeutic, prophylactic, or diagnostic purposes.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straightchains, C₃-C₃₀ for branched chains), preferably 20 or fewer, morepreferably 15 or fewer, most preferably 10 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6, or 7 carbons in the ring structure. The term“alkyl” (or “lower alkyl”) as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving one or more substituents replacing a hydrogen on one or morecarbons of the hydrocarbon backbone. Such substituents include, but arenot limited to, halogen, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester,a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate,phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic orheteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, a “promoter site” is a sequence of nucleotides to whichan RNA polymerase, such as the DNA-dependent RNA polymerase originallyisolated from bacteriophage, described by Davanloo, et al., Proc. Natl.Acad. Sci. USA, 81:2035-39 (1984), or from another source, binds withhigh specificity, as described by Chamberlin, et al., Nature,228:227-231 (1970).

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000, preferablygreater than 64, more preferably greater than 100, most preferablygreater than 300 or 400. poly(A) sequences can be modified chemically orenzymatically to modulate mRNA functionality such as localization,stability or efficiency of translation.

As used herein, an “open reading frame” or “ORF” is a series ofnucleotides that contains a sequence of bases that could potentiallyencode a polypeptide or protein. An open reading frame is locatedbetween the start-code sequence (initiation codon or start codon) andthe stop-codon sequence (termination codon).

The term “construct” refers to a recombinant genetic molecule having oneor more isolated polynucleotide sequences.

The term “expression control sequence” refers to a nucleic acid sequencethat controls and regulates the transcription and/or translation ofanother nucleic acid sequence. Control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and the like. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

As used herein “to reprogram a cell” or “cellular reprogramming” meansto induce a cell to express one or more polypeptides or functionalnucleic acids in an effective amount to change a function of the cell.The function can be any function. For example, an immune cell can beinduced to express a receptor which changes the cell's ability torecognize an antigen or to mediate an immune response; or a somatic cellcan be induced to express a pluripopency marker(s) which candedifferentiate the cell from a somatic state to a pluripotent state(i.e., induced pluripotent stem cell (iPS)).

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. For example, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence, and an organellelocalization sequence operably linked to protein will assist the linkedprotein to be localized at the specific organelle.

A “transgenic organism” as used herein, is any organism, in which one ormore of the cells of the organism contains heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. Suitabletransgenic organisms include, but are not limited to, bacteria,cyanobacteria, fungi, plants and animals. The nucleic acids describedherein can be introduced into the host by methods known in the art, forexample infection, transfection, transformation or transconjugation.Techniques for transferring DNA into such organisms are widely known andprovided in references such as Sambrook, et al. (2000) MolecularCloning: A Laboratory Manual, 3^(rd) ed., vol. 1-3, Cold Spring HarborPress, Plainview N.Y.

As used herein, the term “eukaryote” or “eukaryotic” refers to organismsor cells or tissues derived therefrom belonging to the phylogeneticdomain Eukarya such as animals (e.g., mammals, insects, reptiles, andbirds), ciliates, plants (e.g., monocots, dicots, and algae), fungi,yeasts, flagellates, microsporidia, and protists.

As used herein, the term “non-eukaryotic organism” refers to organismsincluding, but not limited to, organisms of the Eubacteria phylogeneticdomain, such as Escherichia coli, Thermus thermophilus, and Bacillusstearothermophilus, or organisms of the Archaea phylogenetic domain suchas, Methanocaldococcus jannaschii, Methanothermobacterthermautotrophicus, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, and Aeuropyrum pernix.

The term “gene” refers to a DNA sequence that encodes through itstemplate or messenger RNA a sequence of amino acids characteristic of aspecific peptide, polypeptide, or protein. The term “gene” also refersto a DNA sequence that encodes an RNA product. The term gene as usedherein with reference to genomic DNA includes intervening, non-codingregions as well as regulatory regions and can include 5′ and 3′ ends.

The term “orthologous genes” or “orthologs” refer to genes that have asimilar nucleic acid sequence because they were separated by aspeciation event.

The term polypeptide includes proteins and fragments thereof. Thepolypeptides can be “exogenous,” meaning that they are “heterologous,”i.e., foreign to the host cell being utilized, such as human polypeptideproduced by a bacterial cell. Polypeptides are disclosed herein as aminoacid residue sequences. Those sequences are written left to right in thedirection from the amino to the carboxy terminus. In accordance withstandard nomenclature, amino acid residue sequences are denominated byeither a three letter or a single letter code as indicated as follows:Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid(Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

“Variant” refers to a polypeptide or polynucleotide that differs from areference polypeptide or polynucleotide, but retains essentialproperties. A typical variant of a polypeptide differs in amino acidsequence from another, reference polypeptide. Generally, differences arelimited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more modifications (e.g., substitutions, additions, and/ordeletions). A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides which do not significantly alter the characteristics of thepolypeptide (e.g., a conservative amino acid substitution). For example,certain amino acids can be substituted for other amino acids in asequence without appreciable loss of activity. Because it is theinteractive capacity and nature of a polypeptide that defines thatpolypeptide's biological functional activity, certain amino acidsequence substitutions can be made in a polypeptide sequence andnevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, andcofactors. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide ofinterest.

The term “isolated” is meant to describe a compound of interest (e.g.,nucleic acids) that is in an environment different from that in whichthe compound naturally occurs, e.g., separated from its natural milieusuch as by concentrating a peptide to a concentration at which it is notfound in nature. “Isolated” is meant to include compounds that arewithin samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified. Isolated nucleic acids are at least 60% free,preferably 75% free, and most preferably 90% free from other associatedcomponents.

The term “vector” refers to a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors can beexpression vectors.

The term “expression vector” refers to a vector that includes one ormore expression control sequences.

“Transformed,” “transgenic,” “transfected” and “recombinant” refer to ahost organism into which a heterologous nucleic acid molecule has beenintroduced. The nucleic acid molecule can be stably integrated into thegenome of the host or the nucleic acid molecule can also be present asan extrachromosomal molecule. Such an extrachromosomal molecule can beauto-replicating. Transformed cells, tissues, or plants are understoodto encompass not only the end product of a transformation process, butalso transgenic progeny thereof. A “non-transformed,” “non-transgenic,”or “non-recombinant” host refers to a wild-type organism, e.g., a cell,bacterium or plant, which does not contain the heterologous nucleic acidmolecule.

The term “endogenous” with regard to a nucleic acid refers to nucleicacids normally present in the host.

The term “heterologous” refers to elements occurring where they are notnormally found. For example, a promoter may be linked to a heterologousnucleic acid sequence, e.g., a sequence that is not normally foundoperably linked to the promoter. When used herein to describe a promoterelement, heterologous means a promoter element that differs from thatnormally found in the native promoter, either in sequence, species, ornumber. For example, a heterologous control element in a promotersequence may be a control/regulatory element of a different promoteradded to enhance promoter control, or an additional control element ofthe same promoter. The term “heterologous” thus can also encompass“exogenous” and “non-native” elements.

The term “percent (%) sequence identity” is defined as the percentage ofnucleotides or amino acids in a candidate sequence that are identicalwith the nucleotides or amino acids in a reference nucleic acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For purposes herein, the % sequence identity of a given nucleotides oramino acids sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given sequence Cthat has or comprises a certain % sequence identity to, with, or againsta given sequence D) is calculated as follows:100 times the fraction W/Z,where W is the number of nucleotides or amino acids scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides or amino acidsin D. It will be appreciated that where the length of sequence C is notequal to the length of sequence D, the % sequence identity of C to Dwill not equal the % sequence identity of D to C.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially free(at least 60% free, preferably 75% free, and most preferably 90% free)from other components normally associated with the molecule or compoundin a native environment.

Unless otherwise indicated, the disclosure encompasses conventionaltechniques of molecular biology, microbiology, cell biology andrecombinant DNA, which are within the skill of the art. See, e.g.,Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rdedition (2001); Current Protocols In Molecular Biology [(Ausubel, et al.eds., (1987)]; Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995)Current Protocols in Protein Science (John Wiley & Sons, Inc.); theseries Methods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].

II. Polymers

Poly(amine-co-ester)s or poly(amine-co-amides) are described herein. Inone embodiment, the polymer has the formula:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, x, y, and q are independently integers from 1-1000,and Z is O or NR′, wherein R′ is hydrogen, substituted or unsubstitutedalkyl, or substituted or unsubstituted aryl. In particular embodiments,the values of x, y, and q are such that the weight average molecularweight of the polymer is greater than 20,000 Daltons. The polymer canprepared from one or more lactones, one or more amine-diols ortriamines, and one or more diacids or diesters. In those embodimentswhere two or more different lactone, diacid or diester, and/or triamineor amine-diol monomers are used, than the values of n, o, p, and/or mcan be the same or different.

In some embodiments, Z is O.

In some embodiments, Z is O and n is an integer from 1-16, such 4, 10,13, or 14.

In some embodiments, Z is O, n is an integer from 1-16, such 4, 10, 13,or 14, and m is an integer from 1-10, such as 4, 5, 6, 7, or 8.

In some embodiments, Z is O, n is an integer from 1-16, such 4, 10, 13,or 14, m is an integer from 1-10, such as 4, 5, 6, 7, or 8, and o and pare the same integer from 1-6, such 2, 3, or 4.

In some embodiments, Z is O, n is an integer from 1-16, such 4, 10, 13,or 14, m is an integer from 1-10, such as 4, 5, 6, 7, or 8, and R isalkyl, such a methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl,or aryl, such as phenyl.

In certain embodiments, n is 14 (e.g., pentadecalactone, PDL), m is 7(e.g., diethylsebacate, DES), o and p are 2 (e.g.,N-methyldiethanolamine, MDEA). In certain embodiments, n, m, o, and pare as defined above, and PEG is incorporated as a monomer.

In particular embodiments, the values of x, y, and q are such that theweight average molecular weight of the polymer is greater than 20,000Daltons.

The polymer can prepared from one or more substituted or unsubstitutedlactones, one or more substituted or unsubstituted amine-diols (Z═O) ortriamines (Z═NR′), and one or more substituted or unsubstituted diacidsor diesters. In those embodiments where two or more different lactone,diacid or diester, and/or triamine or amine-diol monomers are used, thanthe values of n, o, p, and/or m can be the same or different.

The monomer units can be substituted at one or more positions with oneor more substituents. Exemplary substituents include, but are notlimited to, alkyl groups, cyclic alkyl groups, alkene groups, cyclicalkene groups, alkynes, halogen, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester,a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate,phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, nitro, heterocyclyl, aralkyl, or an aromatic orheteroaromatic moiety.

The polymer is preferably biocompatible. Readily available lactones ofvarious ring sizes are known to possess low toxicity: for example,polyesters prepared from small lactones, such as poly(caprolactone) andpoly(p-dioxanone) are commercially available biomaterials which havebeen used in clinical applications. Large (e.g., C₁₆-C₂₄) lactones andtheir polyester derivatives are natural products that have beenidentified in living organisms, such as bees.

In other embodiments, the polymer is biocompatible and biodegradable.The nucleic acid(s) encapsulated by and/or associated with the particlescan be released through different mechanisms, including diffusion anddegradation of the polymeric matrix. The rate of release can becontrolled by varying the monomer composition of the polymer and thusthe rate of degradation. For example, if simple hydrolysis is theprimary mechanism of degradation, increasing the hydrophobicity of thepolymer may slow the rate of degradation and therefore increase the timeperiod of release. In all case, the polymer composition is selected suchthat an effective amount of nucleic acid(s) is released to achieve thedesired purpose/outcome.

The polymers can further include a block of an alkylene oxide, such aspolyethylene oxide, polypropylene oxide, and/or polyethyleneoxide-co-polypropylene oxide. The structure of a PEG-containing diblockpolymer is shown below:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, x, y, q, and w are independently integers from1-1000, and Z is O or NR′, wherein R′ is hydrogen, substituted orunsubstituted alkyl, or substituted or unsubstituted aryl. In particularembodiments, the values of x, y, q, and w are such that the weightaverage molecular weight of the polymer is greater than 20,000 Daltons.

The structure of a PEG-containing triblock copolymer is shown below:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, x, y, q, and w are independently integers from1-1000, and Z is O or NR′, wherein R′ is hydrogen, substituted orunsubstituted alkyl, or substituted or unsubstituted aryl. In particularembodiments, the values of x, y, q, and w are such that the weightaverage molecular weight of the polymer is greater than 20,000 Daltons.

The blocks of polyalkylene oxide can located at the termini of thepolymer (i.e., by reacting PEG having one hydroxy group blocked, forexample, with a methoxy group), within the polymer backbone (i.e.,neither of the hydroxyl groups are blocked), or combinations thereof.

III. Microparticles Formed from the Polymers

The polymers described above can be used to prepare micro- and/ornanoparticles having encapsulated therein one or more therapeutic,diagnostic, or prophylactic agents. The agent can be encapsulated withinthe particle, dispersed within the polymer matrix that forms theparticle, covalently or non-covalently associated with the surface ofthe particle or combinations thereof.

The agent to be encapsulated and delivered can be a small molecule agent(i.e., non-polymeric agent having a molecular weight less than 2,000,1500, 1,000, 750, or 500 Dalton) or a macromolecule (e.g., an oligomeror polymer) such as proteins, enzymes, peptides, nucleic acids, etc.Suitable small molecule active agents include organic, inorganic, and/ororganometallic compounds. The particles can be used for in vivo and/orin vitro delivery of the agent.

Exemplary therapeutic agents that can be incorporated into the particlesinclude, but are not limited to. tumor antigens, CD4+ T-cell epitopes,cytokines, chemotherapeutic agents, radionuclides, small molecule signaltransduction inhibitors, photothermal antennas, monoclonal antibodies,immunologic danger signaling molecules, other immunotherapeutics,enzymes, antibiotics, antivirals (especially protease inhibitors aloneor in combination with nucleosides for treatment of HIV or Hepatitis Bor C), anti-parasitics (helminths, protozoans), growth factors, growthinhibitors, hormones, hormone antagonists, antibodies and bioactivefragments thereof (including humanized, single chain, and chimericantibodies), antigen and vaccine formulations (including adjuvants),peptide drugs, anti-inflammatories, immunomodulators (including ligandsthat bind to Toll-Like Receptors to activate the innate immune system,molecules that mobilize and optimize the adaptive immune system,molecules that activate or up-regulate the action of cytotoxic Tlymphocytes, natural killer cells and helper T-cells, and molecules thatdeactivate or down-regulate suppressor or regulatory T-cells), agentsthat promote uptake of the particles into cells (including dendriticcells and other antigen-presenting cells), nutraceuticals such asvitamins, and oligonucleotide drugs (including DNA, RNAs, antisense,aptamers, small interfering RNAs, ribozymes, external guide sequencesfor ribonuclease P, and triplex forming agents).

Representative anti-cancer agents include, but are not limited to,alkylating agents (such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine,carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites(such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosinearabinoside, fludarabine, and floxuridine), antimitotics (includingtaxanes such as paclitaxel and decetaxel and vinca alkaloids such asvincristine, vinblastine, vinorelbine, and vindesine), anthracyclines(including doxorubicin, daunorubicin, valrubicin, idarubicin, andepirubicin, as well as actinomycins such as actinomycin D), cytotoxicantibiotics (including mitomycin, plicamycin, and bleomycin),topoisomerase inhibitors (including camptothecins such as camptothecin,irinotecan, and topotecan as well as derivatives of epipodophyllotoxinssuch as amsacrine, etoposide, etoposide phosphate, and teniposide),antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®), other anti-VEGF compounds; thalidomide(THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®);endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors suchas sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib;transforming growth factor-α or transforming growth factor-β inhibitors,and antibodies to the epidermal growth factor receptor such aspanitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

Exemplary immunomodulatory agents include cytokines, xanthines,interleukins, interferons, oligodeoxynucleotides, glucans, growthfactors (e.g., TNF, CSF, GM-CSF and G-CSF), hormones such as estrogens(diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN®(fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA®(medroxyprogesterone acetate)), and corticosteroids (prednisone,dexamethasone, hydrocortisone).

Examples of immunological adjuvants that can be associated with theparticles include, but are not limited to, TLR ligands, C-Type LectinReceptor ligands, NOD-Like Receptor ligands, RLR ligands, and RAGEligands. TLR ligands can include lipopolysaccharide (LPS) andderivatives thereof, as well as lipid A and derivatives there ofincluding, but not limited to, monophosphoryl lipid A (MPL),glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4′-monophosphoryllipid A.

The particles may also include antigens and/or adjuvants (i.e.,molecules enhancing an immune response). Peptide, protein, and DNA basedvaccines may be used to induce immunity to various diseases orconditions. Cell-mediated immunity is needed to detect and destroyvirus-infected cells. Most traditional vaccines (e.g. protein-basedvaccines) can only induce humoral immunity. DNA-based vaccine representsa unique means to vaccinate against a virus or parasite because a DNAbased vaccine can induce both humoral and cell-mediated immunity. Inaddition, DNA based vaccines are potentially safer than traditionalvaccines. DNA vaccines are relatively more stable and morecost-effective for manufacturing and storage. DNA vaccines consist oftwo major components—DNA carriers (or delivery vehicles) and DNAsencoding antigens. DNA carriers protect DNA from degradation, and canfacilitate DNA entry to specific tissues or cells and expression at anefficient level.

Exemplary diagnostic agents include paramagnetic molecules, fluorescentcompounds, magnetic molecules, and radionuclides, x-ray imaging agents,and contrast agents.

In some embodiments, particles produced using the methods described herein contain less than 80%, less then 75%, less than 70%, less than 60%,less than 50% by weight, less than 40% by weight, less than 30% byweight, less than 20% by weight, less than 15% by weight, less than 10%by weight, less than 5% by weight, less than 1% by weight, less than0.5% by weight, or less than 0.1% by weight of the agent. In someembodiments, the agent may be a mixture of pharmaceutically activeagents. The percent loading is dependent on a variety of factors,including the agent to be encapsulated, the polymer used to prepared theparticles, and/or the method used to prepare the particles.

The particles may provide controlled release of the drug. For example,the unaltered particles may provide release of an effective amount ofthe drug over time based on the rate of diffusion of the drug form theparticle and/or the rate of degradation of the polymer. The polymercomposition can be varied to manipulate the degradation behavior of thepolymer and thus the release rate/time of the agent to be delivered.Alternatively, the particle can be coated with one or more materials toprovide controlled release, such as sustained release or delayed releaseof the agent or agents to be delivered.

Sustained release and delayed release materials are well known in theart. Solid esters of fatty acids, which are hydrolyzed by lipases, canbe spray coated onto microparticles or drug particles. Zein is anexample of a naturally water-insoluble protein. It can be coated ontodrug containing microparticles or drug particles by spray coating or bywet granulation techniques. In addition to naturally water-insolublematerials, some substrates of digestive enzymes can be treated withcross-linking procedures, resulting in the formation of non-solublenetworks. Many methods of cross-linking proteins, initiated by bothchemical and physical means, have been reported. One of the most commonmethods to obtain cross-linking is the use of chemical cross-linkingagents. Examples of chemical cross-linking agents include aldehydes(gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, andgenipin. In addition to these cross-linking agents, oxidized and nativesugars have been used to cross-link gelatin. Cross-linking can also beaccomplished using enzymatic means; for example, transglutaminase hasbeen approved as a GRAS substance for cross-linking seafood products.Finally, cross-linking can be initiated by physical means such asthermal treatment, UV irradiation and gamma irradiation.

To produce a coating layer of cross-linked protein surrounding drugcontaining microparticles or drug particles, a water-soluble protein canbe spray coated onto the microparticles and subsequently cross-linked bythe one of the methods described above. Alternatively, drug-containingmicroparticles can be microencapsulated within protein bycoacervation-phase separation (for example, by the addition of salts)and subsequently cross-linked. Some suitable proteins for this purposeinclude gelatin, albumin, casein, and gluten.

Polysaccharides can also be cross-linked to form a water-insolublenetwork. For many polysaccharides, this can be accomplished by reactionwith calcium salts or multivalent cations, which cross-link the mainpolymer chains. Pectin, alginate, dextran, amylose and guar gum aresubject to cross-linking in the presence of multivalent cations.Complexes between oppositely charged polysaccharides can also be formed;pectin and chitosan, for example, can be complexed via electrostaticinteractions.

Controlled release polymers known in the art include acrylic acid andmethacrylic acid copolymers, methyl methacrylate, methyl methacrylatecopolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylicacid), methacrylic acid alkylamine copolymer poly(methyl methacrylate),poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide,poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described in NF XVII asfully polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename Eudragit®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the tradenames Eudragit®RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D and Eudragit®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEudragit® RL30D and 1:40 in Eudragit® RS30D. The mean molecular weightis about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred.The code designations RL (high permeability) and RS (low permeability)refer to the permeability properties of these agents. Eudragit® RL/RSmixtures are insoluble in water and in digestive fluids. However,multiparticulate systems formed to include the same are swellable andpermeable in aqueous solutions and digestive fluids.

The polymers described above such as Eudragit® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% Eudragit®RL, 50% Eudragit®RL and 50% Eudragit®RS, and 10% Eudragit® RL and 90% Eudragit®RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, Eudragit®L.

Other controlled release materials include methyl acrylate-methylmethacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymersinclude, but are not limited to, cellulosic polymers such as methyl andethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

Suitable coating materials for effecting delayed release include, butare not limited to, cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl cellulose acetate succinate,hydroxypropylmethyl cellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, celluloseacetate trimellitate and carboxymethylcellulose sodium; acrylic acidpolymers and copolymers, preferably formed from acrylic acid,methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylateand/or ethyl methacrylate, and other methacrylic resins that arecommercially available under the tradename Eudragit® (Rohm Pharma;Westerstadt, Germany), including Eudragit® L30D-55 and L100-55 (solubleat pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 and above),Eudragit® S (soluble at pH 7.0 and above, as a result of a higher degreeof esterification), and Eudragits® NE, RL and RS (water-insolublepolymers having different degrees of permeability and expandability);vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinylacetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer,and ethylene-vinyl acetate copolymer; enzymatically degradable polymerssuch as azo polymers, pectin, chitosan, amylose and guar gum; zein andshellac.

A. Compositions for Transfection of Polynucleotides

It has been discovered that the gene delivery ability of polycationicpolymers is due to multiple factors, including polymer molecular weight,hydrophobicity, and charge density. Many synthetic polycationicmaterials have been tested as vectors for non-viral gene delivery, butalmost all are ineffective due to their low efficiency or high toxicity.Most polycationic vectors described previously exhibit high chargedensity, which has been considered a major requirement for effective DNAcondensation. As a result, they are able to deliver genes with highefficiency in vitro but are limited for in vivo applications because oftoxicity related to the excessive charge density.

High molecular weight polymers, particularly terpolymers, and methods ofmaking them using enzyme-catalyzed copolymerization of a lactone with adialkyl diester and an amino diol are disclosed. Thesepoly(amine-co-ester) terpolymers have a low charge density. In addition,their hydrophobicity can be varied by selecting a lactone comonomer withspecific ring size and by adjusting lactone content in the polymers.High molecular weight and increased hydrophobicity of thelactone-diester-amino diol terpolymers compensate for the low chargedensity to provide efficient gene delivery with minimal toxicity.

In preferred embodiments, the terpolymers exhibit efficient genedelivery with reduced toxicity. The terpolymers can be significantlymore efficient the commercially available non-viral vectors. Forexamples, the terpolymers described herein can be more than 100× moreefficient than commercially available non-viral vectors such as PEI andLIPOFECTAMINE 2000 based on luciferase expression assay while exhibitingminimal toxicity at doses of up to 0.5 mg/ml toxicity compared to thesecommercially available non-viral vectors. Preferably, the terpolymer isnon-toxic at concentrations suitable for both in vitro and in vivotransfection of nucleic acids. For example, in some embodiments, thedisclosed terpolymers cause less non-specific cell death compared toother approaches of cell transfection.

As described in more detail below, in some embodiments, the terpolymeris ω-pentadecalactone-diethyl sebacate-N-methyldiethanolamine terpolymercontaining 20% PDL (also referred to as terpolymer III-20% PDL).

IV. Micelles Formed from the Polymers

A. Micelle Properties

1. Micelle Size

The polymers described herein, such as PEG-block containing polymers canbe used to prepare micelles. The average micelle size is typically inthe range from about 100 to about 500 nm, preferably from about 100 toabout 400 nm, more preferably from about 100 to about 300 nm, morepreferably from about 150 to about 200 nm, most preferably from about160 to about 190 nm, which were stable at physiological pH of 7.4 in thepresence of serum proteins. The copolymers possess high bloodcompatibility and exhibit minimal activity to induce hemolysis andagglutination.

2. Surface Charge

The size and zeta potential of the micelles were found to changesignificantly when the pH of the aqueous medium accommodating themicelles was varied. For example, the trends in the size-pH and zeta-pHcurves are remarkably similar for the micelles of the three PEG2K-PPMScopolymers with different PDL contents (11%, 30%, and 51%). It isevident that the average size of the micelle samples gradually increasesupon decreasing the medium pH from 7.4 to 5.0, and then remains nearlyconstant when the pH value is below 5.0. This pH-responsive behaviorobserved for the micelles is anticipated since upon decreasing the pHfrom 7.4 to 5.0, the PPMS cores of the micelles become protonated andmore hydrophilic, thus absorbing more water molecules from the aqueousmedium to cause swelling of the micelles. 18 It is assumed that themicelle cores are already fully protonated at pH of 5.0, and as theresult, the sizes of the micelles remain fairly constant with furtherdecreasing of the pH from 5.0. The effects of the PDL content in thePEG2K-PPMS copolymers on the magnitude of the micelle size changebetween 7.4 and 5.0 pH values are also notable. With decreasing PDLcontent and increasing tertiary amino group content in the copolymer,the capacity of the micelle cores to absorb protons and water moleculesis expected to increase. Thus, upon decreasing pH from 7.4 to 5.0, thechange in average micelle size was more significant for PEG2K-PPMS-11%PDL (from 200 nm to 234 nm) as compared to PEG2K-PPMS-30% PDL (from 184nm to 214 nm) and PEG2K-PPMS-51% PDL (from 163 nm to 182 nm) (FIG.4A).40

The zeta potential of the micelles in aqueous medium also exhibitssubstantial pH-dependence. At physiological and alkaline pH (7.4 to8.5), the surface charges of blank PEG2K-PPMS copolymer micelles werenegative, which changed to positive when the pH of the medium decreasedto acidic range (4.0-6.0). For example, the micelles of PEG2K-PPMS-11%PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL possessed zeta potentialvalues of −5.8, −7.1, −5.1 mV, respectively, at pH of 7.4, which turnedto +7.6, +5.8, +4.0 mV, correspondingly, at a lower pH of 5.0. On thebasis of the above discussions, this surface charge dependence on pH isattributable to the protonation or deprotonation of the PPMS cores ofthe micelles at different medium pH. At an alkaline pH (7.4-8.5), mostof the amino groups in the micelles presumably are not protonated, andthe micelle particles remain negatively charged due to the absorption ofHPO42− and/or H2PO4− anions in PBS by the micelles. In particular, at pHof 8.5, the zeta-potential values were −8.1 mV, −7.9 mV, −9.0 mV forPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL,respectively. Upon decreasing pH from 7.4 to 5.0, the tertiary aminomoieties in the micelle PPMS cores become mostly protonated, turning themicelles to positively charged particles. Consistently, among the threemicelle samples, PEG2K-PPMS-11% PDL micelles with the largest capacityto absorb protons displayed the highest zeta potential values at pH of4.0-5.0, whereas PEG2K-PPMS-51% PDL micelles with the smallestprotonation capacity showed the lowest zeta potentials. The observedmicelle surface charge responses to the medium pH are highly desirablesince the negative surface charge of the micelles at physiological pHcan alleviate the interaction of the micelles with serum protein in theblood and prolong their in vivo circulation time. On the other hand, thereverse to positive surface charge at the tumor extracellular pH ofapproximately 6.5 could enhance the uptake of these micelles by targettumor cells.

The surface charge of the particles/micelles were slightly negative inPBS solution (0.01M, pH=7.4), which are beneficial for in vivo drugdelivery applications of the micelles. It is known that nanoparticleswith nearly neutral surface charge (zeta potential between −10 and +10mV) can decrease their uptake by the reticuloendothelial system (RES)and prolong their circulation time in the blood. The negative surfacecharges of the micelles could result from the absorption of HPO₄ ²⁻and/or H₂PO₄ ⁻ anions in PBS by the micelle particles via hydrogenbonding interactions between the anions and the ether groups of PEGshells or the amino groups of PPMS cores. For amphiphilic blockcopolymer micelles, it is anticipated that hydrophilic chain segments(e.g., PEG) in the outer shell of the micelles can shield the charges inthe micelle core with the long chain blocks being more effective inreducing zeta potential than the short chain blocks. Thus, significantlylower zeta potential values were observed for PEG5K-PPMS copolymermicelles as compared to PEG2K-PPMS copolymer micelles.

The copolymer micelles are pH-responsive: decreasing the medium pH from7.4 to 5.0, the sizes of the micelles significantly increased micellesize while the micelle surface charges reversed from negative charges topositive charges. Correspondingly, DTX-encapsulated copolymer micellesshowed gradual sustained drug release at pH of 7.4, but remarkablyaccelerated DTX release at acidic pH of 5.0. This phenomenon can beexploited to improve release of agents at tumor site, since it is knownthat the tumor microenvironment is typically weakly acidic (e.g.,5.7-7.0) as the result of lactic acid accumulation due to poor oxygenperfusion. In contrast, the extracellular pH of the normal tissue andblood is slightly basic (pH of 7.2-7.4). Thus, enhanced drug deliveryefficiency is anticipated for anticancer drug-loaded micelles that arepH-responsive and can be triggered by acidic pH to accelerate the drugrelease. Furthermore, even more acidic conditions (pH=4.0-6.0) areencountered in endosomes and lysosomes after uptake of the micelles bytumor cells via endocytosis pathways, which may further increase thecytotoxicity of the drug-encapsulated micelles.

V. Methods of Making the Polymers

Methods for the synthesis of the polymers from a lactone, a dialkylester, and a dialkyl amine using an enzyme catalyst, such as a lipase,are also provided. Exemplary lactones are shown in FIG. 1. In oneembodiment, the polymers are prepared as shown in Scheme 1:

wherein n is an integer from 1-30, m, o, and p are independently aninteger from 1-20, and x, y, and q are independently integers from1-1000. The polymer can prepared from one or more lactones, one or moreamine-diols or triamines, and one or more diacids or diesters. In thoseembodiments where two or more different lactone, diacid or diester,and/or triamine or amine-diol monomers are used, than the values of n,o, p, and/or m can be the same or different.

The synthesis of the polymers described herein using PDL, DES, MDA, andPEG as reactants is shown in Scheme 2.

The molar ratio of the monomers can vary, for example from about10:90:90 to about 90:10:10. In some embodiments, the ratio is 10:90:90,20:80:80, 40:60:60, 60:40:40, or 80:20:20. The weight average molecularweight, as determined by GPC using narrow polydispersity polystyrenestandards, can vary for example from about 10,000 Daltons to about50,000 Daltons, preferably from about 15,000 Daltons to about 50,000Daltons.

The enzymatic method described herein allows for the synthesis ofpolymers with diverse chain structures and tunable hydrophobicities. Insome embodiments, the hydrophobicity is varied by varying the ring sizeand/or molar amount of the lactone monomer. Lactone with a wide range ofring sizes (e.g., C₄-C₂₄, preferably C₆-C₂₄, more preferably fromC₆-C₁₆) can be used as comonomers. The reaction can be performed in asingle step without protection and deprotection of the amino group(s).Such amino-bearing copolyesters are extremely difficult to prepare usingconventional organometallic catalysts, as such catalysts are oftensensitive to or deactivated by organic amines. These catalysts are alsoknown to be inefficient for polymerizing large lactone ring monomers.Enzymatic catalysts have distinct advantages for producing biomedicalpolymers owing to the high activity and selectivity of the enzyme andthe resulting high purity of products that are metal-free.

Exemplary polymers prepared from a lactone (e.g., caprolactone (CL),ω-pentadecalactone (PDL), 16-hexadecanolide (HDL)), diethyl sebacate(DES), and a dialkyl amine (e.g., N-methyldiethanolamine (MDEA)) aredescribed in Table 1 below. To simplify nomenclature, CL-DES-MDEA,DDL-DES-MDEA, PDL-DES-MDEA, and HDL-DES-MDEA terpolymers are designatedas polymer I, II, III, and IV, respectively.

Table 1 Shows the Yield, Composition, Weight Average Molecular Weight,Polydispersity, and Other Characterization Data of Selected Terpolymers.

Lactone/DES/MDEA Lactone/Sebacate/MDEA Isolated Nitrogen Solubility(Feed Molar (Unit Molar Yield Content in DMSO Name^(a) Ratio) Ratio)^(b)(%) M_(w) ^(c) M_(w)/M_(n) ^(c) (wt %) mg ml⁻¹ PMSC  0:50:50  0:50:50 —31800 2.3 4.9 >25 I- 10:90:90 10:90:90 85 18400 1.9 4.7 >25 10% CL I-20:80:80 20:80:80 80 19100 1.9 4.5 >25 20% CL I- 40:60:60 40:60:60 8318400 1.8 3.9 >25 40% CL I- 60:40:40 60:40:40 81 17800 1.8 3.1 >25 60%CL I- 80:20:20 80:20:20 86 20300 2.0 1.9 >25 80% CL II- 10:90:9010:90:90 82 24900 1.9 4.6 >25 10% DD II- 20:80:80 20:80:80 80 29300 2.04.2 >25 20% DD II- 40:60:60 40:60:60 81 25800 1.8 3.4 >25 40% DD II-60:40:40 60:40:40 84 47400 2.1 2.4 60% DD II- 80:20:20 80:20:20 87 406002.1 1.3 80% DD III- 10:90:90 10:90:90 81 30700 2.1 4.5 >25 10% PD III-20:80:80 20:80:80 83 38700 2.3 4.1 ≈25 20% PD III- 40:60:60 40:60:60 8533300 2.1 3.1 40% PD III- 60:40:40 61:39:39 83 34500 2.3 2.1 61% PD III-80:20:20 82:18:18 88 41700 2.7 1.0 82% PD IV- 10:90:90 10:90:90 80 257001.8 4.5 >25 10% HD IV- 20:80:80 20:80:80 81 26600 1.9 4.0 20% HD IV-40:60:60 40:60:60 83 31200 2.2 3.1 40% HD IV- 60:40:40 61:39:39 86 374002.2 2.0 61% HD IV- 80:20:20 80:20:20 89 59000 2.1 1.1 80% HD

a. The polymer names are abbreviated or simplified. PMSC:poly(N-methyldiethyleneamine sebacate).

Polymers I, II, III, and IV represent CL-DES-MDEA, DDL-DES-MDEA,PDL-DES-MDEA, and HDL-DES-MDEA terpolymers, respectively. Each polymeris denoted with x % lactone indicating the lactone unit content [mol %vs. (lactone+sebacate) units] in the polymer.

b. Measured by ¹H NMR spectroscopy.

c. Measured by GPC using narrow polydispersity polystyrene standards.

C. Therapeutic, Prophylactic and Diagnostic Agents

The polymers described herein can form various polymer compositions,which are useful for preparing a variety of biodegradable medicaldevices and for drug delivery. Devices prepared from the PHA copolymersdescribed herein can be used for a wide range of different medicalapplications. Examples of such applications include controlled releaseof therapeutic, prophylactic or diagnostic agents; drug delivery; tissueengineering scaffolds; cell encapsulation; targeted delivery;biocompatible coatings; biocompatible implants; guided tissueregeneration; wound dressings; orthopedic devices; prosthetics and bonecements (including adhesives and/or structural fillers); anddiagnostics.

The polymers described herein can be used to encapsulate, be mixed with,or be ionically or covalently coupled to any of a variety oftherapeutic, prophylactic or diagnostic agents. A wide variety ofbiologically active materials can be encapsulated or incorporated,either for delivery to a site by the polymer, or to impart properties tothe polymer, such as bioadhesion, cell attachment, enhancement of cellgrowth, inhibition of bacterial growth, and prevention of clotformation.

Examples of suitable therapeutic and prophylactic agents includesynthetic inorganic and organic compounds, proteins and peptides,polysaccharides and other sugars, lipids, and DNA and RNA nucleic acidsequences having therapeutic, prophylactic or diagnostic activities.Nucleic acid sequences include genes, antisense molecules which bind tocomplementary DNA to inhibit transcription, and ribozymes. Compoundswith a wide range of molecular weight can be encapsulated, for example,between 100 and 500,000 grams or more per mole. Examples of suitablematerials include proteins such as antibodies, receptor ligands, andenzymes, peptides such as adhesion peptides, saccharides andpolysaccharides, synthetic organic or inorganic drugs, and nucleicacids. Examples of materials which can be encapsulated include enzymes,blood clotting factors, inhibitors or clot dissolving agents such asstreptokinase and tissue plasminogen activator; antigens forimmunization; hormones and growth factors; polysaccharides such asheparin; oligonucleotides such as antisense oligonucleotides andribozymes and retroviral vectors for use in gene therapy. The polymercan also be used to encapsulate cells and tissues. Representativediagnostic agents are agents detectable by x-ray, fluorescence, magneticresonance imaging, radioactivity, ultrasound, computer tomagraphy (CT)and positron emission tomagraphy (PET). Ultrasound diagnostic agents aretypically a gas such as air, oxygen or perfluorocarbons. In a preferredembodiment, the polymers are used for delivery of nucleic acids.

Polynucleotides

As discussed in more detail below, the disclosed terpolymers can be usedto transfect cells with nucleic acids. Accordingly, polyplexes includingterpolymers and one or more polynucleotides are also disclosed.

The polynucleotide can encode one or more proteins, functional nucleicacids, or combinations thereof. The polynucleotide can be monocistronicor polycistronic. In some embodiments, polynucleotide is multigenic.

In some embodiments, the polynucleotide is transfected into the cell andremains extrachromosomal. In some embodiments, the polynucleotide isintroduced into a host cell and is integrated into the host cell'sgenome. As discussed in more detail below, the compositions can be usedin methods of gene therapy. Methods of gene therapy can include theintroduction into the cell of a polynucleotide that alters the genotypeof the cell. Introduction of the polynucleotide can correct, replace, orotherwise alter the endogenous gene via genetic recombination. Methodscan include introduction of an entire replacement copy of a defectivegene, a heterologous gene, or a small nucleic acid molecule such as anoligonucleotide. For example, a corrective gene can be introduced into anon-specific location within the host's genome.

In some embodiments, the polynucleotide is incorporated into or part ofa vector. Methods to construct expression vectors containing geneticsequences and appropriate transcriptional and translational controlelements are well known in the art. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Expression vectors generally contain regulatory sequencesand necessary elements for the translation and/or transcription of theinserted coding sequence, which can be, for example, the polynucleotideof interest. The coding sequence can be operably linked to a promoterand/or enhancer to help control the expression of the desired geneproduct. Promoters used in biotechnology are of different typesaccording to the intended type of control of gene expression. They canbe generally divided into constitutive promoters, tissue-specific ordevelopment-stage-specific promoters, inducible promoters, and syntheticpromoters.

For example, in some embodiments, the polynucleotide of interest isoperably linked to a promoter or other regulatory elements known in theart. Thus, the polynucleotide can be a vector such as an expressionvector. The engineering of polynucleotides for expression in aprokaryotic or eukaryotic system may be performed by techniquesgenerally known to those of skill in recombinant expression. Anexpression vector typically comprises one of the disclosed compositionsunder the control of one or more promoters. To bring a coding sequence“under the control of” a promoter, one positions the 5′ end of thetranslational initiation site of the reading frame generally betweenabout 1 and 50 nucleotides “downstream” of (i.e., 3′ of) the chosenpromoter. The “upstream” promoter stimulates transcription of theinserted DNA and promotes expression of the encoded recombinant protein.This is the meaning of “recombinant expression” in the context usedhere.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide expression in a variety of host-expression systems.Cell types available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantphage DNA, plasmid DNA or cosmid DNA expression vectors. It will beappreciated that any of these vectors may be packaged and deliveredusing the disclosed polymers.

Expression vectors for use in mammalian cells ordinarily include anorigin of replication (as necessary), a promoter located in front of thegene to be expressed, along with any necessary ribosome binding sites,RNA splice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and may be desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40 (SV40). The early and late promotersof SV40 virus are useful because both are obtained easily from the virusas a fragment which also contains the SV40 viral origin of replication.Smaller or larger SV40 fragments may also be used, provided there isincluded the approximately 250 bp sequence extending from the HindIIIsite toward the BglI site located in the viral origin of replication.

In cases where an adenovirus is used as an expression vector, the codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing proteins in infectedhosts.

Specific initiation signals may also be required for efficienttranslation of the disclosed compositions. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may additionally need to beprovided. One of ordinary skill in the art would readily be capable ofdetermining this need and providing the necessary signals. It is wellknown that the initiation codon must be in-frame (or in-phase) with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements or transcriptionterminators.

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding proteins may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with vectors controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines.

1. Polypeptide of Interest

The polynucleotide can encode one or more polypeptides of interest. Thepolypeptide can be any polypeptide. For example, the polypeptide encodedby the polynucleotide can be a polypeptide that provides a therapeuticor prophylactic effect to an organism or that can be used to diagnose adisease or disorder in an organism. For example, for treatment ofcancer, autoimmune disorders, parasitic, viral, bacterial, fungal orother infections, the polynucleotide(s) to be expressed may encode apolypeptide that functions as a ligand or receptor for cells of theimmune system, or can function to stimulate or inhibit the immune systemof an organism. As discussed in the example below, a polynucleotideencoding TNF-related apoptosis-inducing ligand (TRAIL) can be deliveredto tumor cells using the disclosed polyplexes in a method of treatingcancer.

In some embodiments, the polynucleotide supplements or replaces apolynucleotide that is defective in the organism.

In some embodiments, the polynucleotide includes a selectable marker,for example, a selectable marker that is effective in a eukaryotic cell,such as a drug resistance selection marker. This selectable marker genecan encode a factor necessary for the survival or growth of transformedhost cells grown in a selective culture medium. Typical selection genesencode proteins that confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, kanamycin, gentamycin, Zeocin,or tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients withheld from the media.

In some embodiments, the polynucleotide includes a reporter gene.Reporter genes are typically genes that are not present or expressed inthe host cell. The reporter gene typically encodes a protein whichprovides for some phenotypic change or enzymatic property. Examples ofsuch genes are provided in Weising et al. Ann. Rev. Genetics, 22, 421(1988). Preferred reporter genes include glucuronidase (GUS) gene andGFP genes.

2. Functional Nucleic Acids

The polynucleotide can be, or can encode a functional nucleic acid.Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing non-limiting categories: antisense molecules, siRNA, miRNA,aptamers, ribozymes, triplex forming molecules, RNAi, and external guidesequences. The functional nucleic acid molecules can act as effectors,inhibitors, modulators, and stimulators of a specific activity possessedby a target molecule, or the functional nucleic acid molecules canpossess a de novo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA or the genomic DNA of a targetpolypeptide or they can interact with the polypeptide itself. Oftenfunctional nucleic acids are designed to interact with other nucleicacids based on sequence homology between the target molecule and thefunctional nucleic acid molecule. In other situations, the specificrecognition between the functional nucleic acid molecule and the targetmolecule is not based on sequence homology between the functionalnucleic acid molecule and the target molecule, but rather is based onthe formation of tertiary structure that allows specific recognition totake place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. There are numerous methods foroptimization of antisense efficiency by finding the most accessibleregions of the target molecule. Exemplary methods include in vitroselection experiments and DNA modification studies using DMS and DEPC.It is preferred that antisense molecules bind the target molecule with adissociation constant (K_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰,or 10⁻¹².

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP and theophiline, as well as large molecules, suchas reverse transcriptase and thrombin. Aptamers can bind very tightlywith K_(d)'s from the target molecule of less than 10⁻¹² M. It ispreferred that the aptamers bind the target molecule with a K_(d) lessthan 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamers can bind the target moleculewith a very high degree of specificity. For example, aptamers have beenisolated that have greater than a 10,000 fold difference in bindingaffinities between the target molecule and another molecule that differat only a single position on the molecule. It is preferred that theaptamer have a K_(d) with the target molecule at least 10, 100, 1000,10,000, or 100,000 fold lower than the K_(d) with a background bindingmolecule. It is preferred when doing the comparison for a molecule suchas a polypeptide, that the background molecule be a differentpolypeptide.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly. It ispreferred that the ribozymes catalyze intermolecular reactions. Thereare a number of different types of ribozymes that catalyze nuclease ornucleic acid polymerase type reactions which are based on ribozymesfound in natural systems, such as hammerhead ribozymes. There are also anumber of ribozymes that are not found in natural systems, but whichhave been engineered to catalyze specific reactions de novo. Preferredribozymes cleave RNA or DNA substrates, and more preferably cleave RNAsubstrates. Ribozymes typically cleave nucleic acid substrates throughrecognition and binding of the target substrate with subsequentcleavage. This recognition is often based mostly on canonical ornon-canonical base pair interactions. This property makes ribozymesparticularly good candidates for target specific cleavage of nucleicacids because recognition of the target substrate is based on the targetsubstrates sequence.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed in which there are three strands of DNA forming acomplex dependent on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a K_(d) less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹².

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, which is recognized by RNase P, whichthen cleaves the target molecule. EGSs can be designed to specificallytarget a RNA molecule of choice. RNAse P aids in processing transfer RNA(tRNA) within a cell. Bacterial RNAse P can be recruited to cleavevirtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. Similarly,eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized tocleave desired targets within eukarotic cells. Representative examplesof how to make and use EGS molecules to facilitate cleavage of a varietyof different target molecules are known in the art.

Gene expression can also be effectively silenced in a highly specificmanner through RNA interference (RNAi). This silencing was originallyobserved with the addition of double stranded RNA (dsRNA) (Fire, et al.(1998) Nature, 391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89;Hannon, (2002) Nature, 418:244-51). Once dsRNA enters a cell, it iscleaved by an RNase III-like enzyme, Dicer, into double stranded smallinterfering RNAs (siRNA) 21-23 nucleotides in length that contains 2nucleotide overhangs on the 3′ ends (Elbashir, et al. (2001) Genes Dev.,15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al.(2000) Nature, 404:293-6). In an ATP dependent step, the siRNAs becomeintegrated into a multi-subunit protein complex, commonly known as theRNAi induced silencing complex (RISC), which guides the siRNAs to thetarget RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21). At somepoint the siRNA duplex unwinds, and it appears that the antisense strandremains bound to RISC and directs degradation of the complementary mRNAsequence by a combination of endo and exonucleases (Martinez, et al.(2002) Cell, 110:563-74). However, the effect of iRNA or siRNA or theiruse is not limited to any type of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, an siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs. Sequence specific gene silencing can be achieved inmammalian cells using synthetic, short double-stranded RNAs that mimicthe siRNAs produced by the enzyme dicer (Elbashir, et al. (2001) Nature,411:494 498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can bechemically or in vitro-synthesized or can be the result of shortdouble-stranded hairpin-like RNAs (shRNAs) that are processed intosiRNAs inside the cell. Synthetic siRNAs are generally designed usingalgorithms and a conventional DNA/RNA synthesizer. Suppliers includeAmbion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette,Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg,Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands).siRNA can also be synthesized in vitro using kits such as Ambion'sSILENCER® siRNA Construction Kit.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAse (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors.

3. Composition of the Polynucleotides

The polynucleotide can be DNA or RNA nucleotides which typically includea heterocyclic base (nucleic acid base), a sugar moiety attached to theheterocyclic base, and a phosphate moiety which esterifies a hydroxylfunction of the sugar moiety. The principal naturally-occurringnucleotides comprise uracil, thymine, cytosine, adenine and guanine asthe heterocyclic bases, and ribose or deoxyribose sugar linked byphosphodiester bonds.

The polynucleotide can be composed of nucleotide analogs that have beenchemically modified to improve stability, half-life, or specificity oraffinity for a target sequence, relative to a DNA or RNA counterpart.The chemical modifications include chemical modification of nucleobases,sugar moieties, nucleotide linkages, or combinations thereof. As usedherein ‘modified nucleotide” or “chemically modified nucleotide” definesa nucleotide that has a chemical modification of one or more of theheterocyclic base, sugar moiety or phosphate moiety constituents. Insome embodiments, the charge of the modified nucleotide is reducedcompared to DNA or RNA oligonucleotides of the same nucleobase sequence.For example, the oligonucleotide can have low negative charge, nocharge, or positive charge. Modifications should not prevent, andpreferably enhance, the ability of the oligonucleotides to enter a celland carry out a function such inhibition of gene expression as discussedabove.

Typically, nucleoside analogs support bases capable of hydrogen bondingby Watson-Crick base pairing to standard polynucleotide bases, where theanalog backbone presents the bases in a manner to permit such hydrogenbonding in a sequence-specific fashion between the oligonucleotideanalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). Preferred analogs are thosehaving a substantially uncharged, phosphorus containing backbone.

As discussed in more detail below, in one preferred embodiment, theoligonucleotide is a morpholino oligonucleotide.

a. Heterocyclic Bases

The principal naturally-occurring nucleotides include uracil, thymine,cytosine, adenine and guanine as the heterocyclic bases. Theoligonucleotides can include chemical modifications to their nucleobaseconstituents. Chemical modifications of heterocyclic bases orheterocyclic base analogs may be effective to increase the bindingaffinity or stability in binding a target sequence. Chemically-modifiedheterocyclic bases include, but are not limited to, inosine,5-(1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC),5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5and 2-amino-5-(2′-deoxy-.beta.-D-ribofuranosyl)pyridine(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidinederivatives.

b. Sugar Modifications

Polynucleotides can also contain nucleotides with modified sugarmoieties or sugar moiety analogs. Sugar moiety modifications include,but are not limited to, 2′-O-aminoetoxy, 2′-O-amonioethyl (2′-OAE),2′-O-methoxy, 2′-O-methyl, 2-guanidoethyl (2′-OGE), 2′-O,4′-C-methylene(LNA), 2′-O-(methoxyethyl) (2′-OME) and 2′-O—(N-(methyl)acetamido)(2′-OMA). 2′-O-aminoethyl sugar moiety substitutions are especiallypreferred because they are protonated at neutral pH and thus suppressthe charge repulsion between the TFO and the target duplex. Thismodification stabilizes the C3′-endo conformation of the ribose ordexyribose and also forms a bridge with the i-1 phosphate in the purinestrand of the duplex.

The polynucleotide can be a morpholino oligonucleotide. Morpholinooligonucleotides are typically composed of two more morpholino monomerscontaining purine or pyrimidine base-pairing moieties effective to bind,by base-specific hydrogen bonding, to a base in a polynucleotide, whichare linked together by phosphorus-containing linkages, one to threeatoms long, joining the morpholino nitrogen of one monomer to the 5′exocyclic carbon of an adjacent monomer. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil orthymine. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

Important properties of the morpholino-based subunits typically include:the ability to be linked in a oligomeric form by stable, unchargedbackbone linkages; the ability to support a nucleotide base (e.g.adenine, cytosine, guanine, thymidine, uracil or inosine) such that thepolymer formed can hybridize with a complementary-base target nucleicacid, including target RNA, with high T_(m), even with oligomers asshort as 10-14 bases; the ability of the oligomer to be activelytransported into mammalian cells; and the ability of an oligomer:RNAheteroduplex to resist RNAse degradation. In some embodiments,oligonucleotides employ morpholino-based subunits bearing base-pairingmoieties, joined by uncharged linkages.

c. Internucleotide Linkages

Internucleotide bond refers to a chemical linkage between two nucleosidemoieties. Modifications to the phosphate backbone of DNA or RNAoligonucleotides may increase the binding affinity or stabilitypolynucleotides, or reduce the susceptibility of polynucleotides tonuclease digestion. Cationic modifications, including, but not limitedto, diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP)may be especially useful due to decrease electrostatic repulsion betweenthe oligonucleotide and a target. Modifications of the phosphatebackbone may also include the substitution of a sulfur atom for one ofthe non-bridging oxygens in the phosphodiester linkage. Thissubstitution creates a phosphorothioate internucleoside linkage in placeof the phosphodiester linkage. Oligonucleotides containingphosphorothioate internucleoside linkages have been shown to be morestable in vivo.

Examples of modified nucleotides with reduced charge include modifiedinternucleotide linkages such as phosphate analogs having achiral anduncharged intersubunit linkages (e.g., Sterchak, et al., Organic Chem.,52:4202, (1987)), and uncharged morpholino-based polymers having achiralintersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506), as discussedabove. Some internucleotide linkage analogs include morpholidate,acetal, and polyamide-linked heterocycles.

In another embodiment, the oligonucleotides are composed of lockednucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides(see, for example, Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAsform hybrids with DNA which are more stable than DNA/DNA hybrids, aproperty similar to that of peptide nucleic acid (PNA)/DNA hybrids.Therefore, LNA can be used just as PNA molecules would be. LNA bindingefficiency can be increased in some embodiments by adding positivecharges to it. Commercial nucleic acid synthesizers and standardphosphoramidite chemistry are used to make LNAs.

In some embodiments, the oligonucleotides are composed of peptidenucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics inwhich the phosphate backbone of the oligonucleotide is replaced in itsentirety by repeating N-(2-aminoethyl)-glycine units and phosphodiesterbonds are typically replaced by peptide bonds. The various heterocyclicbases are linked to the backbone by methylene carbonyl bonds. PNAsmaintain spacing of heterocyclic bases that is similar to conventionalDNA oligonucleotides, but are achiral and neutrally charged molecules.Peptide nucleic acids are comprised of peptide nucleic acid monomers.

Other backbone modifications include peptide and amino acid variationsand modifications. Thus, the backbone constituents of oligonucleotidessuch as PNA may be peptide linkages, or alternatively, they may benon-peptide peptide linkages. Examples include acetyl caps, aminospacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein asO-linkers), amino acids such as lysine are particularly useful ifpositive charges are desired in the PNA, and the like. Methods for thechemical assembly of PNAs are well known. See, for example, U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571,and 5,786,571.

Polynucleotides optionally include one or more terminal residues ormodifications at either or both termini to increase stability, and/oraffinity of the oligonucleotide for its target. Commonly used positivelycharged moieties include the amino acids lysine and arginine, althoughother positively charged moieties may also be useful. For example,lysine and arginine residues can be added to a bis-PNA linker or can beadded to the carboxy or the N-terminus of a PNA strand. Polynucleotidesmay further be modified to be end capped to prevent degradation using a3′ propylamine group. Procedures for 3′ or 5′ capping oligonucleotidesare well known in the art.

D. Coating Agents for Polyplexes

Efficiency of polynucleotide delivery using the disclosed polymers canbe affected by the positive charges on the polyplex surface. Forexample, a zeta potential of the polyplex of +8.9 mV can attract andbind with negatively charged plasma proteins in the blood duringcirculation and lead to rapid clearance by the reticuloendothelialsystem (RES). Efficiency can also be affected by instability of thepolyplex nanoparticles. For example, as discussed in the Examples below,polyplex particles incubated in NaAc buffer solution containing 10%serum nearly doubled in size within 15 minutes and increased by over10-fold after 75 minutes. As a result of this increase in size, enlargedpolyplexes might be cleared from the circulation by uptake in the liver.Therefore, in some embodiments the polyplexes are treated or coated toimprove polynucleotide delivery efficiency. In some embodiments, thecoating improves cell specific targeting of the polyplex, improves thestability (i.e., stabilizes the size of the polyplex in vivo), increasesthe half-life of the polyplex in vivo (i.e., in systemic circulation),or combinations thereof compared to a control. In some embodiments, thecontrol is a polyplex without a coating.

1. Compositions for Altering Surface Charge

Polynucleotide delivery efficiency of the disclosed polyplexes can beimproved by coating the particles with an agent that is negativelycharged at physiological pH. Preferably, the negatively charged agent iscapable of electrostatic binding to the positively charged surface ofthe polyplexes. The negatively charged agent can neutralize the chargeof the polyplex, or reverse the charge of the polyplex. Therefore, insome embodiments, the negatively charged agent imparts a net negativecharge to the polyplex.

In some embodiments, the negatively charged agent is a negativelycharged polypeptide. For example, the polypeptide can include asparticacids, glutamic acids, or a combination therefore, such that the overallcharge of the polypeptide is a negative at neutral pH. In someembodiments, the polypeptide is a poly aspartic acid polypeptideconsisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more than 20 aspartic acid residues. In some embodiments, thepolypeptide is a poly glutamic acid polypeptide consisting of 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20glutamic acid residues. Other negatively charged molecules include smallmolecules (i.e., MW less than 1500, 100, 750, or 500 Daltons) such ashyaluronic acid.

Increasing the negative charge on the surface of the particle can reduceor prevent the negative interactions described above, wherein morepositively charged particles attract and bind negatively charged plasmaproteins in the blood during circulation and lead to rapid clearance bythe reticuloendothelial system (RES). In some embodiments, the zetapotential of the particles is from about −15 mV to about 10 mV,preferably from about −15 mV to about 8 mV, more preferably from about−10 mV to about 8 mV, more preferably from about −8 mV to about 8 mV.The zeta potential can be more negative or more positive than the rangesabove provided the particles are stable (i.e., don't aggregate, etc.)and not readily cleared from the blood stream The zeta potential can bemanipulated by coating or functionalizing the particle surface with oneor more moieties which varies the surface charge. Alternatively, themonomers themselves can be functionalized and/or additional monomers canbe introduced into the polymer, which vary the surface charge.

2. Targeting Moieties

In some embodiments, the polyplexes include a cell-type or cell-statespecific targeting domain or targeting signal. Examples of moietieswhich may be linked or unlinked to the polyplexes include, for example,targeting moieties which provide for the delivery of molecules tospecific cells. The targeting signal or sequence can be specific for ahost, tissue, organ, cell, organelle, non-nuclear organelle, or cellularcompartment. For example, the compositions disclosed herein can bemodified with galactosyl-terminating macromolecules to target thecompositions to the liver or to liver cells. The modified compositionsselectively enter hepatocytes after interaction of the carrier galactoseresidues with the asialoglycoprotein receptor present in large amountsand high affinity only on these cells. Moreover, the compositionsdisclosed here can be targeted to other specific intercellular regions,compartments, or cell types.

In one embodiment, the targeting signal binds to its ligand or receptorwhich is located on the surface of a target cell such as to bring thevector and cell membranes sufficiently close to each other to allowpenetration of the vector into the cell. Additional embodiments of thepresent disclosure are directed to specifically deliveringpolynucleotides to specific tissue or cell types, wherein thepolynucleotides can encode a polypeptide or interfere with theexpression of a different polynucleotide. The polynucleotides deliveredto the cell can encode polypeptides that can enhance or contribute tothe functioning of the cell.

The targeting moiety can be an antibody or antigen binding fragmentthereof, an antibody domain, an antigen, a T-cell receptor, a cellsurface receptor, a cell surface adhesion molecule, a majorhistocompatibility locus protein, a viral envelope protein and a peptideselected by phage display that binds specifically to a defined cell.

One skilled in the art will appreciate that the tropism of thepolyplexes described can be altered by merely changing the targetingsignal. It is known in the art that nearly every cell type in a tissuein a mammalian organism possesses some unique cell surface receptor orantigen. Thus, it is possible to incorporate nearly any ligand for thecell surface receptor or antigen as a targeting signal. For example,peptidyl hormones can be used a targeting moieties to target delivery tothose cells which possess receptors for such hormones. Chemokines andcytokines can similarly be employed as targeting signals to targetdelivery of the complex to their target cells. A variety of technologieshave been developed to identify genes that are preferentially expressedin certain cells or cell states and one of skill in the art can employsuch technology to identify targeting signals which are preferentiallyor uniquely expressed on the target tissue of interest.

Tumor Targeting

In one embodiment, the targeting signal is used to selectively targettumor cells. Tumor cells express cell surface markers which may only beexpressed in the tumor or present in non-tumor cells but preferentiallypresented in tumor cells. Such markers can be targeted to increasedelivery of the polyplexes to cancer cells.

For example, in some embodiments, the targeting moiety is a polypeptideincluding an arginine-glycine-aspartic acid sequence. For example, thetargeting moiety can be an arginine-glycine-aspartic acid-lysine (RGDK,mRGD) other polypeptide that includes the RGD sequence and is capable ofbinding to tumor endothelium through the interaction of RGD with α_(v)β₃and α_(v)β₅. In some embodiments, a targeting moiety includes thepolypeptide sequence R/KxxR/K, where “x” is any amino acid, and whichallows binding to neuropilin-1. Binding with integrins or neuropilin-1are two approaches for improving tumor-targeted and tissue-penetratingdelivery to tumors in vivo. Similar approaches have been reported tofacilitate ligand-specific gene delivery in vitro and targeted genedelivery to liver, spleen, and bone marrow in vivo.

Other, exemplary tumor specific cell surface markers include, but arenot limited to, alfa-fetoprotein (AFP), C-reactive protein (CRP), cancerantigen-50 (CA-50), cancer antigen-125 (CA-125) associated with ovariancancer, cancer antigen 15-3 (CA15-3) associated with breast cancer,cancer antigen-19 (CA-19) and cancer antigen-242 associated withgastrointestinal cancers, carcinoembryonic antigen (CEA), carcinomaassociated antigen (CAA), chromogranin A, epithelial mucin antigen(MC5), human epithelium specific antigen (HEA), Lewis(a)antigen,melanoma antigen, melanoma associated antigens 100, 25, and 150,mucin-like carcinoma-associated antigen, multidrug resistance relatedprotein (MRPm6), multidrug resistance related protein (MRP41), Neuoncogene protein (C-erbB-2), neuron specific enolase (NSE),P-glycoprotein (mdr1 gene product), multidrug-resistance-relatedantigen, p170, multidrug-resistance-related antigen, prostate specificantigen (PSA), CD56, NCAM, EGFR, CD44, and folate receptor. In oneembodiment, the targeting signal consists of antibodies which arespecific to the tumor cell surface markers.

Antibodies

Another embodiment provides an antibody or antigen binding fragmentthereof bound to the disclosed polyplex acts as the targeting signal.The antibodies or antigen binding fragment thereof are useful fordirecting the polyplex to a cell type or cell state. In one embodiment,the polyplex is coated with a polypeptide that is an antibody bindingdomain, for example from a protein known to bind antibodies such asProtein A and Protein G from Staphylococcus aureus. Other domains knownto bind antibodies are known in the art and can be substituted. Theantibody binding domain links the antibody, or antigen binding fragmentthereof, to the polyplex.

In certain embodiments, the antibody that serves as the targeting signalis polyclonal, monoclonal, linear, humanized, chimeric or a fragmentthereof. Representative antibody fragments are those fragments that bindthe antibody binding portion of the non-viral vector and include Fab,Fab′, F(ab′), Fv diabodies, linear antibodies, single chain antibodiesand bispecific antibodies known in the art.

In some embodiments, the targeting signal includes all or part of anantibody that directs the polyplex to the desired target cell type orcell state. Antibodies can be monoclonal or polyclonal, but arepreferably monoclonal. For human gene therapy purposes, antibodies canbe derived from human genes and are specific for cell surface markers,and are produced to reduce potential immunogenicity to a human host asis known in the art. For example, transgenic mice which contain theentire human immunoglobulin gene cluster are capable of producing“human” antibodies can be utilized. In one embodiment, fragments of suchhuman antibodies are employed as targeting signals. In a preferredembodiment, single chain antibodies modeled on human antibodies areprepared in prokaryotic culture.

Brain Targeting

In one embodiment, the targeting signal is directed to cells of thenervous system, including the brain and peripheral nervous system. Cellsin the brain include several types and states and possess unique cellsurface molecules specific for the type. Furthermore, cell types andstates can be further characterized and grouped by the presentation ofcommon cell surface molecules.

In one embodiment, the targeting signal is directed to specificneurotransmitter receptors expressed on the surface of cells of thenervous system. The distribution of neurotransmitter receptors is wellknown in the art and one so skilled can direct the compositionsdescribed by using neurotransmitter receptor specific antibodies astargeting signals. Furthermore, given the tropism of neurotransmittersfor their receptors, in one embodiment the targeting signal consists ofa neurotransmitter or ligand capable of specifically binding to aneurotransmitter receptor.

In one embodiment, the targeting signal is specific to cells of thenervous system which may include astrocytes, microglia, neurons,oligodendrites and Schwann cells. These cells can be further divided bytheir function, location, shape, neurotransmitter class and pathologicalstate. Cells of the nervous system can also be identified by their stateof differentiation, for example stem cells Exemplary markers specificfor these cell types and states are well known in the art and include,but are not limited to CD133 and Neurosphere.

Muscle Targeting

In one embodiment, the targeting signal is directed to cells of themusculoskeletal system. Muscle cells include several types and possessunique cell surface molecules specific for the type and state.Furthermore, cell types and states can be further characterized andgrouped by the presentation of common cell surface molecules.

In one embodiment, the targeting signal is directed to specificneurotransmitter receptors expressed on the surface of muscle cells. Thedistribution of neurotransmitter receptors is well known in the art andone so skilled can direct the compositions described by usingneurotransmitter receptor specific antibodies as targeting signals.Furthermore, given the tropism of neurotransmitters for their receptors,in one embodiment the targeting signal consists of a neurotransmitter.Exemplary neurotransmitters expressed on muscle cells that can betargeted include but are not limited to acetycholine and norepinephrine.

In one embodiment, the targeting signal is specific to muscle cellswhich consist of two major groupings, Type I and Type II. These cellscan be further divided by their function, location, shape, myoglobincontent and pathological state. Muscle cells can also be identified bytheir state of differentiation, for example muscle stem cells. Exemplarymarkers specific for these cell types and states are well known in theart include, but are not limited to MyoD, Pax7, and MR4.

3. Linkers

In some embodiments the polyplex can be coated with both a negativelycharged agent and a targeting moiety. In some embodiments, thenegatively charged agent and the targeting moiety are linked together bya linker. The linker can be a polypeptide, or any other suitable linkerthat is known in the art, for example, poly ethylene glycol (PEG).

In some embodiments, the linker is polypeptide that has approximatelyneutral charge at physiological pH. In some embodiments, the linkerpolypeptide is a polyglycine. For example, in some embodiments thelinker consists of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or glycine residues. In a preferred embodiment, the linkeris a 6-residue polyglycine.

In some embodiments, the negatively charged agent alone, or incombination with a targeting moiety is linked to the polyplex byelectrostatic interactions. In some embodiments, the negative chargedagent, the targeting moiety, or a combination thereof is linked to thepolyplex by covalent conjugation to the polymer backbone or to a sidechain attached to the polymer backbone.

4. Exemplary Polyplex Coating

An exemplary polyplex coating for targeting tumor cells is polyE-mRGD.As used herein, polyE-mRGD refers to a synthetic peptide containingthree segments: a first segment including a polyglutamic acid (polyE)stretch, which is negatively charged at physiological pH and, therefore,capable of electrostatic binding to the positively charged surface ofthe polyplexes; a second segment including a neutral polyglycine strech,which serves as a neutral linker; and a third segment that includes aRGD sequence that binds the tumor endothelium through the interaction ofRGD with α_(v)β₃ and α_(v)β₅.

As discussed in more detail below, the polyE-mRGD used in the Examplesreversed the surface charge of III-20% PDL/pLucDNA polyplex. WhenpolyE-mRGD was added at 5:1 peptide/DNA weight ratio, the zeta potentialof the polyplex changed from +8.9 mV to −5.8 mV. Peptide coatedpolyplexes were stable upon incubation in NaAc buffer containing 10%serum and resistant to aggregation indicating that the modifiedpolyplexes can escape clearance by RES during circulation in vivo.

In one embodiment, polyE-mRGD includes the sequenceEEEEEEEEEEEEEEEEGGGGGGRGDK (SEQ ID NO:1), or RGDKGGGGGG EEEEEEEEEEEEEEEE(SEQ ID NO:2), or a variant thereof with 85%, 90%, 95%, or more than 95%sequence identity to SEQ ID NO:1 or 2.

Another exemplary coating that can be used to prepare charge neutral, ornegatively charged particles that maintain their size in vivo aredescribed in Harris, et al., Biomaterials, 31:998-1006 (2010)), and caninclude the amino acid sequence GGGGGGEEEEEEEEEEEEEEEE (SEQ ID NO:3,poly-E), for non-specific systemic administration, or the amino acidssequence GdPdLGdVdRG-GGGGGG-EEEEEEEEEEEEEEEE-CONH2 (SEQ ID NO:4,poly-E-cat), which contains a polycationic sequence that increasetargeting to the spleen, spine, sternum, and femur. In some embodiments,the polypeptide used in the coating is a variant SEQ ID NO:3 or 4, with85%, 90%, 95%, or more than 95% sequence identity to SEQ ID NO:3 or 4

In vitro studies have indicated that adsorption of immunoglobulin G(IgG) and complement protein C3 to nanoparticles increases their uptakeby Kupffer cells and incubation in serum increases hepatic uptake invivo following liver perfusion (Nagayama, et al., Int. J. Pharm.,342:215-21 (2007)). Reports also indicate that galactose can be used toguide polymeric gene delivery particles to hepatocytes via theasialoglycoprotein receptor (ASGPR (SEQ ID NO:6) (Zhang, et al., J.Controlled Release, 102:749-63 (2005)).

E. Size of Polyplexes and Methods of Reducing Aggregation

Resistance to aggregation can be important because maintaining a smallparticle size limits clearance by the liver and maintains transfectionability of polyplex particles into target cells. Therefore, in preferredembodiments, the polyplexes are resistant to aggregation. Preferably,polyplexes with or without coating are between about 1 nm and 1000 nm inradius, more preferably between about 1 nm and about 500 nm in radius,most preferably between about 15 nm and about 250 nm in radius. Forexample, in some embodiments, coated polyplexes loaded withpolynucleotide are between about 150 nm and 275 nm in radius.

The ratio of polynucleotide weight to polymer weight(polynucletide:polymer), the content and quantity of polyplex coating,or a combination thereof can be used to adjust the size of thepolyplexes.

For example, the Examples below show that in some embodiments,transfection efficiency of particles with 25:1 polymer to DNA ratio islower than the transfection efficiency of particles with 50:1, 100:1,150:1, and 200:1 polymer:DNA ratios. The most preferredpolymer:polynucleotide ratio for a particular formulation can bedetermined empirically using the methods that are known in the art, suchas those described in the Examples below. Generally, the weight:weightratio of polymer:polynucleotide is preferably greater than about 10:1,more preferably greater than about 50:1, most preferably greater thanabout 100:1. The weight:weight ratio of polymer:polynucleotide ispreferably between about 10:1 and 500:1, more preferably between about25:1 and 250:1, most preferably between about 50:1 and 150:1. In someembodiments, the weight ratio of polymer:polynucleotide is about 100:1.Preferably, the polyplexes has are spherical in shape.

Examples below also show that in some embodiments, transfectionefficiency of particles by the ratio of coating agent molecules topolynucleotide molecules (coating agent:polynucleotide). The ratio isexpressed by weight. The most preferred coating agent:polynucleotideratio for a particular formulation can be determined empirically usingthe methods that are known in the art, such as those described in theExamples below. Generally, the ratio of coating agent:polynucleotide isgreater than 0, and preferably lower than about 50:1, more preferablylower than about 25:1, most preferably lower than about 10:1. The ratiocoating agent:polynucleotide is preferably between about 1:1 and 10:1,more preferably between about 2.5:1 and 7.5:1. In some embodiments, theratio of coating agent:polynucleotide is about 5:1. Ratios of coatingagent:polynucleotide of 10:1, 5:1, and 2.5:1 are also referred to hereinas 10×, 5×, and 2.5× respectively. Preferably, the polyplexes arespherical in shape.

F. Formulations

Formulations are prepared using a pharmaceutically acceptable “carrier”composed of materials that are considered safe and effective and may beadministered to an individual without causing undesirable biologicalside effects or unwanted interactions. The “carrier” is all componentspresent in the pharmaceutical formulation other than the activeingredient or ingredients. The term “carrier” includes but is notlimited to diluents, binders, lubricants, desintegrators, fillers, andcoating compositions.

“Carrier” also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. The delayed release dosage formulations may be prepared asdescribed in references such as “Pharmaceutical dosage form tablets”,eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989),“Remington—The science and practice of pharmacy”, 20th ed., LippincottWilliams & Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosageforms and drug delivery systems”, 6^(th) Edition, Ansel et. al., (Media,Pa.: Williams and Wilkins, 1995) which provides information on carriers,materials, equipment and process for preparing tablets and capsules anddelayed release dosage forms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name Eudragit®(Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also termed “fillers,” aretypically necessary to increase the bulk of a solid dosage form so thata practical size is provided for compression of tablets or formation ofbeads and granules. Suitable diluents include, but are not limited to,dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose,mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin,sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch,silicone dioxide, titanium oxide, magnesium aluminum silicate and powdersugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads granules or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, and preservatives.

As will be appreciated by those skilled in the art and as described inthe pertinent texts and literature, a number of methods are availablefor preparing drug-containing tablets, beads, granules or particles thatprovide a variety of drug release profiles. Such methods include, butare not limited to, the following: coating a drug or drug-containingcomposition with an appropriate coating material, typically although notnecessarily incorporating a polymeric material, increasing drug particlesize, placing the drug within a matrix, and forming complexes of thedrug with a suitable complexing agent.

The delayed release dosage units may be coated with the delayed releasepolymer coating using conventional techniques, e.g., using aconventional coating pan, an airless spray technique, fluidized bedcoating equipment (with or without a Wurster insert), or the like. Fordetailed information concerning materials, equipment and processes forpreparing tablets and delayed release dosage forms, see PharmaceuticalDosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker,Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, 6.sup.th Ed. (Media, Pa.: Williams & Wilkins, 1995).

A preferred method for preparing extended release tablets is bycompressing a drug-containing blend, e.g., blend of granules, preparedusing a direct blend, wet-granulation, or dry-granulation process.Extended release tablets may also be molded rather than compressed,starting with a moist material containing a suitable water-solublelubricant. However, tablets are preferably manufactured usingcompression rather than molding. A preferred method for forming extendedrelease drug-containing blend is to mix drug particles directly with oneor more excipients such as diluents (or fillers), binders,disintegrants, lubricants, glidants, and colorants. As an alternative todirect blending, a drug-containing blend may be prepared by usingwet-granulation or dry-granulation processes. Beads containing theactive agent may also be prepared by any one of a number of conventionaltechniques, typically starting from a fluid dispersion. For example, atypical method for preparing drug-containing beads involves dispersingor dissolving the active agent in a coating suspension or solutioncontaining pharmaceutical excipients such as polyvinylpyrrolidone,methylcellulose, talc, metallic stearates, silicone dioxide,plasticizers or the like. The admixture is used to coat a bead core suchas a sugar sphere (or so-called “non-pareil”) having a size ofapproximately 60 to 20 mesh.

An alternative procedure for preparing drug beads is by blending drugwith one or more pharmaceutically acceptable excipients, such asmicrocrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone,talc, magnesium stearate, a disintegrant, etc., extruding the blend,spheronizing the extrudate, drying and optionally coating to form theimmediate release beads.

Extended Release Dosage Forms

The extended release formulations are generally prepared as diffusion orosmotic systems, for example, as described in “Remington—The science andpractice of pharmacy” (20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000). A diffusion system typically consists of twotypes of devices, reservoir and matrix, and is well known and describedin the art. The matrix devices are generally prepared by compressing thedrug with a slowly dissolving polymer carrier into a tablet form. Thethree major types of materials used in the preparation of matrix devicesare insoluble plastics, hydrophilic polymers, and fatty compounds.Plastic matrices include, but not limited to, methyl acrylate-methylmethacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymersinclude, but are not limited to, methylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and carbopol 934, polyethylene oxides. Fattycompounds include, but are not limited to, various waxes such ascarnauba wax and glyceryl tristearate.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above couldbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include multilayer tablets, capsulescontaining tablets, beads, granules, etc.

An immediate release portion can be added to the extended release systemby means of either applying an immediate release layer on top of theextended release core using coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation processes. Their formulations usuallyincorporate polymers, diluents, binders, and lubricants as well as theactive pharmaceutical ingredient. The usual diluents include inertpowdered substances such as any of many different kinds of starch,powdered cellulose, especially crystalline and microcrystallinecellulose, sugars such as fructose, mannitol and sucrose, grain floursand similar edible powders. Typical diluents include, for example,various types of starch, lactose, mannitol, kaolin, calcium phosphate orsulfate, inorganic salts such as sodium chloride and powdered sugar.Powdered cellulose derivatives are also useful. Typical tablet bindersinclude substances such as starch, gelatin and sugars such as lactose,fructose, and glucose. Natural and synthetic gums, including acacia,alginates, methylcellulose, and polyvinylpyrrolidine can also be used.Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes canalso serve as binders. A lubricant is necessary in a tablet formulationto prevent the tablet and punches from sticking in the die. Thelubricant is chosen from such slippery solids as talc, magnesium andcalcium stearate, stearic acid and hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In a congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

Delayed Release Dosage Forms

Delayed release formulations are created by coating a solid dosage formwith a film of a polymer which is insoluble in the acid environment ofthe stomach, and soluble in the neutral environment of small intestines.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit®. (RohmPharma; Westerstadt, Germany), including Eudragit®. L30D-55 and L100-55(soluble at pH 5.5 and above), Eudragit®. L-100 (soluble at pH 6.0 andabove), Eudragit®. S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and Eudragits®. NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

Pulsatile Release Formulations

By “pulsatile” is meant that a plurality of drug doses are released atspaced apart intervals of time. Generally, upon ingestion of the dosageform, release of the initial dose is substantially immediate, i.e., thefirst drug release “pulse” occurs within about one hour of ingestion.This initial pulse is followed by a first time interval (lag time)during which very little or no drug is released from the dosage form,after which a second dose is then released. Similarly, a second nearlydrug release-free interval between the second and third drug releasepulses may be designed. The duration of the nearly drug release-freetime interval will vary depending upon the dosage form design e.g., atwice daily dosing profile, a three times daily dosing profile, etc. Fordosage forms providing a twice daily dosage profile, the nearly drugrelease-free interval has a duration of approximately 3 hours to 14hours between the first and second dose. For dosage forms providing athree times daily profile, the nearly drug release-free interval has aduration of approximately 2 hours to 8 hours between each of the threedoses.

In one embodiment, the pulsatile release profile is achieved with dosageforms that are closed and preferably sealed capsules housing at leasttwo drug-containing “dosage units” wherein each dosage unit within thecapsule provides a different drug release profile. Control of thedelayed release dosage unit(s) is accomplished by a controlled releasepolymer coating on the dosage unit, or by incorporation of the activeagent in a controlled release polymer matrix. Each dosage unit maycomprise a compressed or molded tablet, wherein each tablet within thecapsule provides a different drug release profile. For dosage formsmimicking a twice a day dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, whilea second tablet releases drug approximately 3 hours to less than 14hours following ingestion of the dosage form. For dosage forms mimickinga three times daily dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, asecond tablet releases drug approximately 3 hours to less than 10 hoursfollowing ingestion of the dosage form, and the third tablet releasesdrug at least 5 hours to approximately 18 hours following ingestion ofthe dosage form. It is possible that the dosage form includes more thanthree tablets. While the dosage form will not generally include morethan a third tablet, dosage forms housing more than three tablets can beutilized.

Alternatively, each dosage unit in the capsule may comprise a pluralityof drug-containing beads, granules or particles. As is known in the art,drug-containing “beads” refer to beads made with drug and one or moreexcipients or polymers. Drug-containing beads can be produced byapplying drug to an inert support, e.g., inert sugar beads coated withdrug or by creating a “core” comprising both drug and one or moreexcipients. As is also known, drug-containing “granules” and “particles”comprise drug particles that may or may not include one or moreadditional excipients or polymers. In contrast to drug-containing beads,granules and particles do not contain an inert support. Granulesgenerally comprise drug particles and require further processing.Generally, particles are smaller than granules, and are not furtherprocessed. Although beads, granules and particles may be formulated toprovide immediate release, beads and granules are generally employed toprovide delayed release.

For dosage forms mimicking a twice a day dosing profile, a first groupof beads, granules or particles releases drug substantially immediatelyfollowing ingestion of the dosage form, while a second group of beads orgranules preferably releases drug approximately 3 hours to less than 14hours following ingestion of the dosage form. For dosage forms mimickinga three times daily dosing profile, a first group of beads, granules orparticles releases drug substantially immediately following ingestion ofthe dosage form, a second group of beads or granules preferably releasesdrug approximately 3 hours to 10 hours following ingestion of the dosageform, and a third group of beads, granules or particles releases drug atleast 5 hours to approximately 18 hours following ingestion of thedosage form. The above-mentioned tablets, beads, granules or particlesof different drug release profiles (e.g., immediate and delayed releaseprofiles) may be mixed and included in a capsule, tablet or matrix toprovide a pulsatile dosage form having the desired release profile.

In another embodiment, the individual dosage units are compacted in asingle tablet, and may represent integral but discrete segments thereof(e.g., layers), or may be present as a simple admixture. For example,drug-containing beads, granules or particles with different drug releaseprofiles (e.g., immediate and delayed release profiles) can becompressed together into a single tablet using conventional tabletingmeans.

In a further alternative embodiment, a dosage form is provided thatcomprises an inner drug-containing core and at least one drug-containinglayer surrounding the inner core. An outer layer of this dosage formcontains an initial, immediate release dose of the drug. For dosageforms mimicking twice daily dosing, the dosage form has an outer layerthat releases drug substantially immediately following oraladministration and an inner core having a polymeric-coating thatpreferably releases the active agent approximately 3 hours to less than14 hours following ingestion of the dosage unit. For dosage formsmimicking three times daily dosing, the dosage form has an outer layerthat releases drug substantially immediately following oraladministration, an inner core that preferably releases drug at least 5hours to 18 hours following oral administration and a layer interposedbetween the inner core and outer layer that preferably releases drugapproximately 3 hours to 10 hours following ingestion of the dosageform. The inner core of the dosage form mimicking three times dailydosing may be formulated as compressed delayed release beads orgranules.

Alternatively, for dosage forms mimicking three times daily dosing, thedosage form has an outer layer and an inner layer free of drug. Theouter layer releases drug substantially immediately following oraladministration, and completely surrounds the inner layer. The innerlayer surrounds both the second and third doses and preferably preventsrelease of these doses for approximately 3 hours to 10 hours followingoral administration. Once released, the second dose is immediatelyavailable while the third dose is formulated as delayed release beads orgranules such that release of the third dose is effected approximately 2hours to 8 hours thereafter effectively resulting in release of thethird dose at least 5 hours to approximately 18 hours followingingestion of the dosage form. The second and third doses may beformulated by admixing immediate release and delayed release beads,granules or particles and compressing the admixture to form a second andthird dose-containing core followed by coating the core with a polymercoating to achieve the desired three times daily dosing profile.

In still another embodiment, a dosage form is provided which comprises acoated core-type delivery system wherein the outer layer is comprised ofan immediate release dosage unit containing an active agent, such thatthe active agent therein is immediately released following oraladministration; an intermediate layer there under which surrounds acore; and a core which is comprised of immediate release beads orgranules and delayed release beads or granules, such that the seconddose is provided by the immediate release beads or granules and thethird dose is provided by the delayed release beads or granules.

As will be appreciated by those skilled in the art and as described inthe pertinent texts and literature, a number of methods are availablefor preparing drug-containing tablets, beads, granules or particles thatprovide a variety of drug release profiles. Such methods include, butare not limited to, the following: coating a drug or drug-containingcomposition with an appropriate coating material, typically although notnecessarily incorporating a polymeric material; increasing drug particlesize; placing the drug within a matrix; and forming complexes of thedrug with suitable complexing agents.

Exemplary methods of preparing polyplexes for transfection are discussedin the Examples below.

VI. Methods of Preparing Polyplexes

A. Methods for Making Particles

Particles can be prepared using a variety of techniques known in theart. The technique to be used can depend on a variety of factorsincluding the polymer used to form the nanoparticles, the desired sizerange of the resulting particles, and suitability for the material to beencapsulated. Suitable techniques include, but are not limited to:

a. Solvent Evaporation. In this method the polymer is dissolved in avolatile organic solvent. The drug (either soluble or dispersed as fineparticles) is added to the solution, and the mixture is suspended in anaqueous solution that contains a surface active agent such as poly(vinylalcohol). The resulting emulsion is stirred until most of the organicsolvent evaporated, leaving solid nanoparticles. The resultingnanoparticles are washed with water and dried overnight in alyophilizer. Nanoparticles with different sizes and morphologies can beobtained by this method.

b. Hot Melt Microencapsulation. In this method, the polymer is firstmelted and then mixed with the solid particles. The mixture is suspendedin a non-miscible solvent (like silicon oil), and, with continuousstirring, heated to 5° C. above the melting point of the polymer. Oncethe emulsion is stabilized, it is cooled until the polymer particlessolidify. The resulting nanoparticles are washed by decantation withpetroleum ether to give a free-flowing powder. The external surfaces ofspheres prepared with this technique are usually smooth and dense.

c. Solvent Removal. In this method, the drug is dispersed or dissolvedin a solution of the selected polymer in a volatile organic solvent.This mixture is suspended by stirring in an organic oil (such as siliconoil) to form an emulsion. Unlike solvent evaporation, this method can beused to make nanoparticles from polymers with high melting points anddifferent molecular weights. The external morphology of spheres producedwith this technique is highly dependent on the type of polymer used.

d. Spray-Drying. In this method, the polymer is dissolved in organicsolvent. A known amount of the active drug is suspended (insolubledrugs) or co-dissolved (soluble drugs) in the polymer solution. Thesolution or the dispersion is then spray-dried.

e. Phase Inversion. Nanospheres can be formed from polymers using aphase inversion method wherein a polymer is dissolved in a “good”solvent, fine particles of a substance to be incorporated, such as adrug, are mixed or dissolved in the polymer solution, and the mixture ispoured into a strong non solvent for the polymer, to spontaneouslyproduce, under favorable conditions, polymeric microspheres, wherein thepolymer is either coated with the particles or the particles aredispersed in the polymer. The method can be used to producenanoparticles in a wide range of sizes, including, for example, about100 nanometers to about 10 microns. Substances which can be incorporatedinclude, for example, imaging agents such as fluorescent dyes, orbiologically active molecules such as proteins or nucleic acids. In theprocess, the polymer is dissolved in an organic solvent and thencontacted with a non solvent, which causes phase inversion of thedissolved polymer to form small spherical particles, with a narrow sizedistribution optionally incorporating an antigen or other substance.

Other methods known in the art that can be used to prepare nanoparticlesinclude, but are not limited to, polyelectrolyte condensation (see Suket al., Biomaterials, 27, 5143-5150 (2006)); single and double emulsion(probe sonication); nanoparticle molding, and electrostaticself-assembly (e.g., polyethylene imine-DNA or liposomes).

In one embodiment, the loaded particles are prepared by combining asolution of the polymer, typically in an organic solvent, with thepolynucleotide of interest. The polymer solution is prepared bydissolving or suspending the polymer in a solvent. The solvent should beselected so that it does not adversely effect (e.g., destabilize ordegrade) the nucleic acid to be encapsulated. Suitable solvents include,but are not limited to DMSO and methylene chloride. The concentration ofthe polymer in the solvent can be varied as needed. In some embodiments,the concentration is for example 25 mg/ml. The polymer solution can alsobe diluted in a buffer, for example, sodium acetate buffer.

Next, the polymer solution is mixed with the agent to be encapsulated,such as a polynucleotide. The agent can be dissolved in a solvent toform a solution before combining it with the polymer solution. In someembodiments, the agent is dissolved in a physiological buffer beforecombining it with the polymer solution. The ratio of polymer solutionvolume to agent solution volume can be 1:1. The combination of polymerand agent are typically incubated for a few minutes to form particlesbefore using the solution for its desired purpose, such as transfection.For example, a polymer/polynucleotide solution can be incubated for 2,5, 10, or more than 10 minutes before using the solution fortransfection. The incubation can be at room temperature.

In some embodiments, the particles are also incubated with a solutioncontaining a coating agent prior to use. The particle solution can beincubated with the coating agent for 2, 5, 10, or more than 10 minutesbefore using the polyplexes for transfection. The incubation can be atroom temperature.

In some embodiments, if the agent is a polynucleotide, thepolynucleotide is first complexed to a polycation before mixing withpolymer. Complexation can be achieved by mixing the polynucleotides andpolycations at an appropriate molar ratio. When a polyamine is used asthe polycation species, it is useful to determine the molar ratio of thepolyamine nitrogen to the polynucleotide phosphate (N/P ratio). In apreferred embodiment, inhibitory RNAs and polyamines are mixed togetherto form a complex at an N/P ratio of between approximately 1:1 to 1:25,preferably between about 8:1 to 15:1. The volume of polyamine solutionrequired to achieve particular molar ratios can be determined accordingto the following formula:

$V_{{NH}\; 2} = \frac{C_{{inhRNA},{final}} \times {M_{w,{inhRNA}}/C_{{inhRNA},{final}}} \times M_{w,P} \times \Phi_{N:P} \times \Phi\; V_{final}}{C_{{NH}\; 2}/M_{w,{{NH}\; 2}}}$where M_(w,inhRNA)=molecular weight of inhibitory RNA, M_(w,P)=molecularweight of phosphate groups of inhibitory RNA, Φ_(N:P)=N:P ratio (molarratio of nitrogens from polyamine to the ratio of phosphates from theinhibitory RNA), C_(NH2), stock=concentration of polyamine stocksolution, and M_(w,NH2)=molecular weight per nitrogen of polyamine.Methods of mixing polynucleotides with polycations to condense thepolynucleotide are known in the art. See for example U.S. PublishedApplication No. 2011/0008451.

The term “polycation” refers to a compound having a positive charge,preferably at least 2 positive charges, at a selected pH, preferablyphysiological pH. Polycationic moieties have between about 2 to about 15positive charges, preferably between about 2 to about 12 positivecharges, and more preferably between about 2 to about 8 positive chargesat selected pH values. Many polycations are known in the art. Suitableconstituents of polycations include basic amino acids and theirderivatives such as arginine, asparagine, glutamine, lysine andhistidine; cationic dendrimers; and amino polysaccharides. Suitablepolycations can be linear, such as linear tetralysine, branched ordendrimeric in structure.

Exemplary polycations include, but are not limited to, syntheticpolycations based on acrylamide and2-acrylamido-2-methylpropanetrimethylamine,poly(N-ethyl-4-vinylpyridine) or similar quartemized polypyridine,diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate,lipopolyamines, poly(allylamines) such as the strong polycationpoly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene,and polypeptides such as protamine, the histone polypeptides,polylysine, polyarginine and polyornithine.

In some embodiments, the polycation is a polyamine. Polyamines arecompounds having two or more primary amine groups. Suitable naturallyoccurring polyamines include, but are not limited to, spermine,spermidine, cadaverine and putrescine. In a preferred embodiment, thepolyamine is spermidine.

In another embodiment, the polycation is a cyclic polyamine. Cyclicpolyamines are known in the art and are described, for example, in U.S.Pat. No. 5,698,546, WO 1993/012096 and WO 2002/010142. Exemplary cyclicpolyamines include, but are not limited to, cyclen.

Spermine and spermidine are derivatives of putrescine(1,4-diaminobutane) which is produced from L-ornithine by action of ODC(ornithine decarboxylase). L-ornithine is the product of L-argininedegradation by arginase. Spermidine is a triamine structure that isproduced by spermidine synthase (SpdS) which catalyzes monoalkylation ofputrescine (1,4-diaminobutane) with decarboxylated S-adenosylmethionine(dcAdoMet) 3-aminopropyl donor. The formal alkylation of both aminogroups of putrescine with the 3-aminopropyl donor yields the symmetricaltetraamine spermine. The biosynthesis of spermine proceeds to spermidineby the effect of spermine synthase (SpmS) in the presence of dcAdoMet.The 3-aminopropyl donor (dcAdoMet) is derived from S-adenosylmethionineby sequential transformation of L-methionine by methionineadenosyltransferase followed by decarboxylation by AdoMetDC(S-adenosylmethionine decarboxylase). Hence, putrescine, spermidine andspermine are metabolites derived from the amino acids L-arginine(L-ornithine, putrescine) and L-methionine (dcAdoMet, aminopropyldonor).

B. Methods for Transfection

Transfection is carried out by contacting cells with the solutioncontaining the polyplexes. For in vivo methods, the contacting typicallyoccurs in vivo after the solution is administered to the subject. For invitro methods, the solution is typically added to a culture of cells andallowed to contact the cells for minutes, hours, or days. The cells cansubsequently be washed to move excess polyplexes.

V. Methods of Using the Particles/micelles

A. Drug delivery

The particles described herein can be use to deliver an effective amountof one or more therapeutic, diagnostic, and/or prophylactic agents to apatient in need of such treatment. The amount of agent to beadministered can be readily determine by the prescribing physician andis dependent on the age and weight of the patient and the disease ordisorder to be treated.

The particles are useful in drug delivery (as used herein “drug”includes therapeutic, nutritional, diagnostic and prophylactic agents),whether injected intravenously, subcutaneously, or intramuscularly,administered to the nasal or pulmonary system, injected into a tumormilieu, administered to a mucosal surface (vaginal, rectal, buccal,sublingual), or encapsulated for oral delivery. The particles may beadministered as a dry powder, as an aqueous suspension (in water,saline, buffered saline, etc), in a hydrogel, organogel, or liposome, incapsules, tablets, troches, or other standard pharmaceutical excipient.

B. Transfection

The disclosed compositions can be for cell transfection ofpolynucleotides. As discussed in more detail below, the transfection canoccur in vitro or in vivo, and can be applied in applications includinggene therapy and disease treatment. The compositions can be moreefficient, less toxic, or a combination thereof when compared to acontrol. In some embodiments, the control is cells treated with analternative transfection reagent such as LIPOFECTAMINE 2000 orpolyethylenimine (PEI).

The particular polynucleotide delivered by the polyplex can be selectedby one of skill in the art depending on the condition or disease to betreated. The polynucleotide can be, for example, a gene or cDNA ofinterest, a functional nucleic acid such as an inhibitory RNA, a tRNA,an rRNA, or an expression vector encoding a gene or cDNA of interest, afunctional nucleic acid a tRNA, or an rRNA. In some embodiments two ormore polynucleotides are administered in combination.

In some embodiments, the polynucleotide encodes a protein. Exemplaryproteins include, for example, (a) angiogenic and other factorsincluding growth factors such as acidic and basic fibroblast growthfactors, vascular endothelial growth factor, endothelial mitogenicgrowth factors, epidermal growth factor, transforming growth factor αand β, platelet-derived endothelial growth factor, platelet-derivedgrowth factor, tumor necrosis factor-α, hepatocyte growth factor andinsulin-like growth factor; (b) cell cycle inhibitors such ascyclin-dependent kinases, thymidine kinase (“TK”), and other agentsuseful for interfering with cell proliferation; (c) bone morphogenicproteins (“BMP's”), including BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1),BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, and BMP-16. BMPs are typically dimeric proteins that can beprovided as homodimers, heterodimers, or combinations thereof, alone ortogether with other molecules. Alternatively, or in addition, moleculescapable of inducing an upstream or downstream effect of a BMP can beprovided. Such molecules include any of the “hedgehog” proteins, or theDNA's encoding them.

In some embodiments, the polynucleotide is not integrated into the hostcell's genome (i.e., remains extrachromosomal). Such embodiments can beuseful for transient or regulated expression of the polynucleotide, andreduce the risk of insertional mutagenesis. Therefore, in someembodiments, the polyplexes are used to deliver mRNA or non-integratingexpression vectors that are expressed transiently in the host cell.

In a preferred embodiment, the polynucleotide is a pro-apoptoticconstruct, for example an expression vector encoding TNF-relatedapoptosis-inducing ligand (TRAIL), which is targeted to tumor cells.

In some embodiments, the polynucleotide is integrated into the hostcell's genome. For example, gene therapy is a technique for correctingdefective genes responsible for disease development. Researchers may useone of several approaches for correcting faulty genes: (a) a normal genecan be inserted into a nonspecific location within the genome to replacea nonfunctional gene. This approach is most common; (b) an abnormal genecan be swapped for a normal gene through homologous recombination; (c)an abnormal gene can be repaired through selective reverse mutation,which returns the gene to its normal function; (d) the regulation (thedegree to which a gene is turned on or off) of a particular gene can bealtered.

Gene therapy can include the use of viral vectors, for example,adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neuronal trophic virus, Sindbis and other RNAviruses, including these viruses with the HIV backbone. Also useful areany viral families which share the properties of these viruses whichmake them suitable for use as vectors. Typically, viral vectors contain,nonstructural early genes, structural late genes, an RNA polymerase IIItranscript, inverted terminal repeats necessary for replication andencapsidation, and promoters to control the transcription andreplication of the viral genome. When engineered as vectors, virusestypically have one or more of the early genes removed and a gene orgene/promoter cassette is inserted into the viral genome in place of theremoved viral DNA.

Gene targeting via target recombination, such as homologousrecombination (HR), is another strategy for gene correction. Genecorrection at a target locus can be mediated by donor DNA fragmentshomologous to the target gene (Hu, et al., Mol. Biotech., 29:197-210(2005); Olsen, et al., J. Gene Med., 7:1534-1544 (2005)). One method oftargeted recombination includes the use of triplex-formingoligonucleotides (TFOs) which bind as third strands tohomopurine/homopyrimidine sites in duplex DNA in a sequence-specificmanner. Triplex forming oligonucleotides can interact with eitherdouble-stranded or single-stranded nucleic acids.

Methods for targeted gene therapy using triplex-forming oligonucleotides(TFO's) and peptide nucleic acids (PNAs) are described in U.S. PublishedApplication No. 20070219122 and their use for treating infectiousdiseases such as HIV are described in U.S. Published Application No.2008050920. The triplex-forming molecules can also be tail clamp peptidenucleic acids (tcPNAs), such as those described in U.S. PublishedApplication No. 2011/0262406. Highly stable PNA:DNA:PNA triplexstructures can be formed from strand invasion of a duplex DNA with twoPNA strands. In this complex, the PNA/DNA/PNA triple helix portion andthe PNA/DNA duplex portion both produce displacement of thepyrimidine-rich triple helix, creating an altered structure that hasbeen shown to strongly provoke the nucleotide excision repair pathwayand to activate the site for recombination with the donoroligonucleotide. Two PNA strands can also be linked together to form abis-PNA molecule.

The triplex-forming molecules are useful to induce site-specifichomologous recombination in mammalian cells when used in combinationwith one or more donor oligonucleotides which provides the correctedsequence. Donor oligonucleotides can be tethered to triplex-formingmolecules or can be separate from the triplex-forming molecules. Thedonor oligonucleotides can contain at least one nucleotide mutation,insertion or deletion relative to the target duplex DNA.

Double duplex-forming molecules, such as a pair of pseudocomplementaryoligonucleotides, can also induce recombination with a donoroligonucleotide at a chromosomal site. Use of pseudocomplementaryoligonucleotides in targeted gene therapy is described in U.S. PublishedApplication No. 2011/0262406. Pseudocomplementary oligonucleotides arecomplementary oligonucleotides that contain one or more modificationssuch that they do not recognize or hybridize to each other, for exampledue to steric hindrance, but each can recognize and hybridize tocomplementary nucleic acid strands at the target site. In someembodiments, pseudocomplementary oligonucleotides are pseudocomplemenarypeptide nucleic acids (pcPNAs). Pseudocomplementary oligonucleotides canbe more efficient and provide increased target site flexibility overmethods of induced recombination such as triple-helix oligonucleotidesand bis-peptide nucleic acids which require a polypurine sequence in thetarget double-stranded DNA.

A. In Vivo Methods

The disclosed compositions can be used in a method of deliveringpolynucleotides to cells in vivo. It has been discovered that thedisclosed polymers are more efficient and/or less toxic for systemic invivo transfection of polynucleotides than alternative transfectionreagents includes LIPOFECTAMINE 2000, PEI, and even other PMSCs.Accordingly, in some embodiments, the cell specific polyplexes includinga therapeutic polynucleotide are administered systemically in vivo to atreat a disease, for example cancer.

In some in vivo approaches, the compositions are administered to asubject in a therapeutically effective amount. As used herein the term“effective amount” or “therapeutically effective amount” means a dosagesufficient to treat, inhibit, or alleviate one or more symptoms of thedisorder being treated or to otherwise provide a desired pharmacologicand/or physiologic effect. The precise dosage will vary according to avariety of factors such as subject-dependent variables (e.g., age,immune system health, etc.), the disease, and the treatment beingeffected.

1. Pharmaceutical Compositions

Pharmaceutical compositions including nucleic acids and, optionally,polypeptides are provided. Pharmaceutical compositions can be foradministration by parenteral (intramuscular, intraperitoneal,intravenous (IV) or subcutaneous injection), transdermal (eitherpassively or using iontophoresis or electroporation), or transmucosal(nasal, vaginal, rectal, or sublingual) routes of administration orusing bioerodible inserts and can be formulated in dosage formsappropriate for each route of administration.

In some embodiments, the compositions are administered systemically, forexample, by intravenous or intraperitoneal administration, in an amounteffective for delivery of the compositions to targeted cells. Otherpossible routes include trans-dermal or oral.

In certain embodiments, the compositions are administered locally, forexample by injection directly into a site to be treated. In someembodiments, the compositions are injected or otherwise administereddirectly to one or more tumors. Typically, local injection causes anincreased localized concentration of the compositions which is greaterthan that which can be achieved by systemic administration. In someembodiments, the compositions are delivered locally to the appropriatecells by using a catheter or syringe. Other means of delivering suchcompositions locally to cells include using infusion pumps (for example,from Alza Corporation, Palo Alto, Calif.) or incorporating thecompositions into polymeric implants (see, for example, P. Johnson andJ. G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England:Ellis Horwood Ltd., 1987), which can effect a sustained release of thepolyplexes to the immediate area of the implant.

The polyplexes can be provided to the cell either directly, such as bycontacting it with the cell, or indirectly, such as through the actionof any biological process. For example, the polyplexes can be formulatedin a physiologically acceptable carrier or vehicle, and injected into atissue or fluid surrounding the cell. The polyplexes can cross the cellmembrane by simple diffusion, endocytosis, or by any active or passivetransport mechanism.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, age, and general health of the recipient, will be able toascertain proper dosing. The selected dosage depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment desired. Generally dosage levels of 0.001 to 10 mg/kgof body weight daily are administered to mammals. Generally, forintravenous injection or infusion, dosage may be lower. Generally, thetotal amount of the polyplex-associated nucleic acid administered to anindividual will be less than the amount of the unassociated nucleic acidthat must be administered for the same desired or intended effect.

2. Formulations for Parenteral Administration

In a preferred embodiment the polyplexes are administered in an aqueoussolution, by parenteral injection. As discussed in the Examples below,in some embodiments, a formulation suitable for systemic administrationby injection includes glucose.

The formulation can be in the form of a suspension or emulsion. Ingeneral, pharmaceutical compositions are provided including effectiveamounts of nucleic acids optionally include pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents sterile water, bufferedsaline of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; and optionally, additives such as detergents andsolubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to aspolysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

3. Formulations for Topical and Mucosal Administration

The polyplexes can be applied topically. Topical administration caninclude application to the lungs, nasal, oral (sublingual, buccal),vaginal, or rectal mucosa.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent® nebulizer(Mallinckrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (MarquestMedical Products, Englewood, Colo.); the Ventolin® metered dose inhaler(Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powderinhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkindall have inhalable insulin powder preparations approved or in clinicaltrials where the technology could be applied to the formulationsdescribed herein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets, capsules, or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations can includepenetration enhancers.

4. Co-Administration

Polyplexes disclosed herein can optionally be co-administered with oneor more additional active agents. Co-administration can include thesimultaneous and/or sequential administration of the one or moreadditional active agents and the polyplexes. The one or more additionalactive agents and the polyplexes can be included in the same ordifferent pharmaceutical formulation. The one or more additional activeagents and the polyplexes can achieve the same or different clinicalbenefit. An appropriate time course for sequential administration may bechosen by the physician, according to such factors as the nature of apatient's illness, and the patient's condition. In certain embodiments,sequential administration includes the co-administration of one or moreadditional active agents and the nanoparticle gene carriers within aperiod of one week, 72 hours, 48 hours, 24 hours, or 12 hours.

The additional active agent can be chosen by the user based on thecondition or disease to be treated. Example of additional active agentsinclude, but are not limited to, vitamin supplements, nutritionalsupplements, anti-anxiety medication, anti-depression medication,anti-coagulants, clotting factors, anti-inflammatories, steroids such ascorticosteroids, analgesic, etc.

If the disease to be treated is cancer, the polyplexes can beadministered to a subject in combination with a chemotherapeutic regime,a radiological treatment, a surgical intervention, or combinationsthereof. For example, in some methods, the polyplexes areco-administered with a chemotherapeutic drug or immunostimulatory drug.The disclosed compositions can be administered with an antibody orantigen binding fragment thereof specific for a growth factor receptorsor tumor specific antigens. Representative growth factors receptorsinclude, but are not limited to, epidermal growth factor receptor (EGFR;HER1); c-erbB2 (HER2); c-erbB3 (HER3); c-erbB4 (HER4); insulin receptor;insulin-like growth factor receptor 1 (IGF-1R); insulin-like growthfactor receptor 2/Mannose-6-phosphate receptor (IGF-II R/M-6-Preceptor); insulin receptor related kinase (IRRK); platelet-derivedgrowth factor receptor (PDGFR); colony-stimulating factor-1 receptor(CSF-1R) (c-Fms); steel receptor (c-Kit); Flk2/Flt3; fibroblast growthfactor receptor 1 (Flg/Cek1); fibroblast growth factor receptor 2(Bek/Cek3/K-Sam); Fibroblast growth factor receptor 3; Fibroblast growthfactor eceptor 4; nerve growth factor receptor (NGFR) (TrkA); BDNFreceptor (TrkB); NT-3-receptor (TrkC); vascular endothelial growthfactor receptor 1 (Flt1); vascular endothelial growth factor receptor2/Flk1/KDR; hepatocyte growth factor receptor (HGF-R/Met); Eph; Eck;Eek; Cek4/Mek4/HEK; Cek5; Elk/Cek6; Cek7; Sek/Cek8; Cek9; Cek10; HEK11;9 Ror1; Ror2; Ret; Ax1; RYK; DDR; and Tie.

Additional therapeutic agents include conventional cancer therapeuticssuch as chemotherapeutic agents, cytokines, chemokines, and radiationtherapy. The majority of chemotherapeutic drugs can be divided in to:alkylating agents, antimetabolites, anthracyclines, plant alkaloids,topoisomerase inhibitors, and other antitumour agents. All of thesedrugs affect cell division or DNA synthesis and function in some way.Additional therapeutics include monoclonal antibodies and the newtyrosine kinase inhibitors e.g. imatinib mesylate (GLEEVEC® or GLIVEC®),which directly targets a molecular abnormality in certain types ofcancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited tocisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxoland derivatives thereof, irinotecan, topotecan, amsacrine, etoposide,etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab(HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®),bevacizumab (AVASTIN®), and combinations thereof.

Other agents that can be administered in combination with polyplexesinclude PD-1 antagonists such as an anti-B7-H1 antibody or an anti-PD-1antibody, an anti-CTLA4 antibody, a mitosis inhibitor, such aspaclitaxel, an aromatase inhibitor, such as letrozole, an A2ARantagonist, an angiogenesis inhibitor, anthracyclines, oxaliplatin,doxorubicin, TLR4 antagonists, and IL-18 antagonists.

B. In vitro Methods

The disclosed compositions can be used in a method of deliveringpolynucleotides to cells in vitro. For example, the polyplexes can beused for in vitro transfection of cells. The method typically involvescontacting the cells with polyplex including a polynucleotide in aneffective amount to introduce the polynucleotide into the cell'scytoplasm. In some embodiments, the polynucleotide is delivered to thecell in an effective amount to change the genotype or a phenotype of thecell. The cells can primary cells isolated from a subject, or cells ofan established cell line. The cells can be of a homogenous cell type, orcan be a heterogeneous mixture of different cells types. For example,the polyplexes can be introduced into the cytoplasm of cells from aheterogenous cell line possessing cells of different types, such as in afeeder cell culture, or a mixed culture in various states ofdifferentiation. The cells can be a transformed cell line that can bemaintained indefinitely in cell culture. Exemplary cell lines are thoseavailable from American Type Culture Collection including tumor celllines.

Any eukaryotic cell can be transfected to produce cells that express aspecific nucleic acid, for example a metabolic gene, including primarycells as well as established cell lines. Suitable types of cells includebut are not limited to undifferentiated or partially differentiatedcells including stem cells, totipotent cells, pluripotent cells,embryonic stem cells, inner mass cells, adult stem cells, bone marrowcells, cells from umbilical cord blood, and cells derived from ectoderm,mesoderm, or endoderm. Suitable differentiated cells include somaticcells, neuronal cells, skeletal muscle, smooth muscle, pancreatic cells,liver cells, and cardiac cells. In another embodiment, siRNA, antisensepolynucleotides (including siRNA or antisense polynucleotides) orinhibitory RNA can be transfected into a cell using the compositionsdescribed herein.

The methods are particularly useful in the field of personalizedtherapy, for example, to repair a defective gene, de-differentiatecells, or reprogram cells. For example, target cells are first isolatedfrom a donor using methods known in the art, contacted with thepolyplexes including a polynucleotide causing a change to the in vitro(ex vivo), and administered to a patient in need thereof. Sources orcells include cells harvested directly from the patient or anallographic donor. In preferred embodiments, the target cells to beadministered to a subject will be autologous, e.g. derived from thesubject, or syngeneic. Allogeneic cells can also be isolated fromantigenically matched, genetically unrelated donors (identified througha national registry), or by using target cells obtained or derived froma genetically related sibling or parent.

Cells can be selected by positive and/or negative selection techniques.For example, antibodies binding a particular cell surface protein may beconjugated to magnetic beads and immunogenic procedures utilized torecover the desired cell type. It may be desirable to enrich the targetcells prior to transient transfection. As used herein in the context ofcompositions enriched for a particular target cell, “enriched” indicatesa proportion of a desirable element (e.g. the target cell) which ishigher than that found in the natural source of the cells. A compositionof cells may be enriched over a natural source of the cells by at leastone order of magnitude, preferably two or three orders, and morepreferably 10, 100, 200, or 1000 orders of magnitude. Once target cellshave been isolated, they may be propagated by growing in suitable mediumaccording to established methods known in the art. Established celllines may also be useful in for the methods. The cells can be storedfrozen before transfection, if necessary.

Next the cells are contacted with the disclosed composition in vitro torepair, de-differentiate, re-differentiate, and/or re-program the cell.The cells can be monitored, and the desired cell type can be selectedfor therapeutic administration.

Following repair, de-differentiation, and/or re-differentiation and/orreprogramming, the cells are administered to a patient in need thereof.In the most preferred embodiments, the cells are isolated from andadministered back to the same patient. In alternative embodiments, thecells are isolated from one patient, and administered to a secondpatient. The method can also be used to produce frozen stocks of alteredcells which can be stored long-term, for later use. In one embodiment,fibroblasts, keratinocytes or hematopoietic stem cells are isolated froma patient and repaired, de-differentiated, or reprogrammed in vitro toprovide therapeutic cells for the patient.

C. Diseases to be Treated

Embodiments of the present disclosure provide compositions and methodsapplicable for gene therapy protocols and the treatment of gene relateddiseases or disorders. Cell dysfunction can also be treated or reducedusing the disclosed compositions and methods. In some embodiments,diseases amenable to gene therapy are specifically targeted. The diseasecan be in children, for example individuals less than 18 years of age,typically less than 12 years of age, or adults, for example individuals18 years of age or more. Thus, embodiments of the present disclosure aredirected to treating a host diagnosed with a disease, by transfection ofthe polyplex including a polynucleotide into the cell affected by thedisease and wherein the polynucleotide encodes a therapeutic protein. Inanother embodiment, an inhibitory RNA is directed to a specific celltype or state to reduce or eliminate the expression of a protein,thereby achieving a therapeutic effect. The present disclosureencompasses manipulating, augmenting or replacing genes to treatdiseases caused by genetic defects or abnormalities.

Suitable genetic based diseases that can be treated with thecompositions disclosed herein include but are not limited to:

Mitochondrial Disease:

Alpers Disease; Barth syndrome; β-oxidation defects;carnitine-acyl-carnitine deficiency; carnitine deficiency; co-enzyme Q10deficiency; Complex I deficiency; Complex II deficiency; Complex IIIdeficiency; Complex IV deficiency; Complex V deficiency; cytochrome coxidase (COX) deficiency, LHON—Leber Hereditary Optic Neuropathy;MM—Mitochondrial Myopathy; LIMM—Lethal Infantile Mitochondrial Myopathy;MMC—Maternal Myopathy and Cardiomyopathy; NARP—Neurogenic muscleweakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease; FICP—FatalInfantile Cardiomyopathy Plus, a MELAS—associated cardiomyopathy;MELAS—Mitochondrial Encephalomyopathy with Lactic Acidosis andStrokelike episodes; LDYT—Leber's hereditary optic neuropathy andDystonia; MERRF—Myoclonic Epilepsy and Ragged Red Muscle Fibers;MHCM—Maternally inherited Hypertrophic CardioMyopathy; CPEO—ChronicProgressive External Ophthalmoplegia; KSS—Kearns Sayre Syndrome;DM—Diabetes Mellitus; DMDF Diabetes Mellitus+DeaFness; CIPO—ChronicIntestinal Pseudoobstruction with myopathy and Ophthalmoplegia;DEAF—Maternally inherited DEAFness or aminoglycoside-induced DEAFness;PEM—Progressive encephalopathy; SNHL—SensoriNeural Hearing Loss;Encephalomyopathy; Mitochondrial cytopathy; Dilated Cardiomyopathy;GER—Gastrointestinal Reflux; DEMCHO—Dementia and Chorea; AMDF—Ataxia,Myoclonus; Exercise Intolerance; ESOC Epilepsy, Strokes, Optic atrophy,& Cognitive decline; FBSN Familial Bilateral Striatal Necrosis; FSGSFocal Segmental Glomerulosclerosis; LIMM Lethal Infantile MitochondrialMyopathy; MDM Myopathy and Diabetes Mellitus; MEPR Myoclonic Epilepsyand Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCMMaternally Inherited Hypertrophic CardioMyopathy; MICM MaternallyInherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome;Mitochondrial Encephalocardiomyopathy; Multisystem MitochondrialDisorder (myopathy, encephalopathy, blindness, hearing loss, peripheralneuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy;NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM ProgressiveEncephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome;SIDS Sudden Infant Death Syndrome; MIDD Maternally Inherited Diabetesand Deafness; and MODY Maturity-Onset Diabetes of the Young.

Nuclear Disease:

Muscular Dystrophies, Ellis-van Creveld syndrome, Marfan syndrome,Myotonic dystrophy, Spinal muscular atrophy, Achondroplasia, Amyotrophiclateral sclerosis, Charcot-Marie-Tooth syndrome, Cockayne syndrome,Diastrophic dysplasia, Duchenne muscular dystrophy, Ellis-van Creveldsyndrome, Fibrodysplasia ossificans progressive, Alzheimer disease,Angelman syndrome, Epilepsy, Essential tremor, Fragile X syndrome,Friedreich's ataxia, Huntington disease, Niemann-Pick disease, Parkinsondisease, Prader-Willi syndrome, Rett syndrome, Spinocerebellar atrophy,Williams syndrome, Ataxia telangiectasia, Anemia, sickle cell, Burkittlymphoma, Gaucher disease, Hemophilia, Leukemia, Paroxysmal nocturnalhemoglobinuria, Porphyria, Thalassemia, Crohn's disease,Alpha-1-antitrypsin deficiency, Cystic fibrosis, Deafness, Pendredsyndrome, Glaucoma, Gyrate atrophy of the choroid and retina, Adrenalhyperplasia, Adrenoleukodystrophy, Cockayne syndrome, Long QT syndrome,Immunodeficiency with hyper-IgM, Alport syndrome, Ellis-van Creveldsyndrome, Fibrodysplasia ossificans progressive, Waardenburg syndrome,Werner syndrome.

Infectious Disease:

Viral—AIDS, AIDS Related Complex, Chickenpox (Varicella), Common cold,Cytomegalovirus Infection, Colorado tick fever, Dengue fever, Ebolahaemorrhagic fever, Epidemic parotitis, Flu, Hand, foot and mouthdisease, Hepatitis—Herpes simplex, Herpes zoster, HPV, Influenza, Lassafever, Measles, Marburg haemorrhagic fever, Infectious mononucleosis,Mumps, Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies,Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viralgastroenteritis, Viral meningitis, Viral pneumonia, West Niledisease—Yellow fever; Bacterial—Anthrax, Bacterial Meningitis,Brucellosis, Bubonic plague, Campylobacteriosis, Cat Scratch Disease,Cholera, Diphtheria, Epidemic Typhus, Gonorrhea, Hansen's Disease,Legionellosis, Leprosy, Leptospirosis, Listeriosis, Lyme Disease,Melioidosis, MRSA infection, Nocardiosis, Pertussis, Pneumococcalpneumonia, Psittacosis, Q fever, Rocky Mountain Spotted Fever or RMSF,Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus, Trachoma,Tuberculosis, Tularemia, Typhoid Fever, Typhus, Whooping Cough;Parasitic—African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis,Chagas Disease, Clonorchiasis, Cryptosporidiosis, Cysticercosis,Diphyllobothriasis, Dracunculiasis, Echinococcosis, Enterobiasis,Fascioliasis, Fasciolopsiasis, Filariasis, Free-living amebic infection,Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Kala-azar,Leishmaniasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis,Pediculosis, Pinworm Infection, Scabies, Schistosomiasis, Taeniasis,Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,Trypanosomiasis.

Cancers:

Breast and ovarian cancer, Burkitt lymphoma, Chronic myeloid leukemia,Colon cancer, Lung cancer, Malignant melanoma, Multiple endocrineneoplasia, Neurofibromatosis, p53 LieFrauMeni, Pancreatic cancer,Prostate cancer, retinoblastoma, von Hippel-Lindau syndrome, Polycystickidney disease, Tuberous sclerosis.

Metabolic Disorders:

Adrenoleukodystrophy, Atherosclerosis, Best disease, Gaucher disease,Glucose galactose malabsorption, Gyrate atrophy, Juvenile onsetdiabetes, Obesity, Paroxysmal nocturnal hemoglobinuria, Phenylketonuria,Refsum disease, Tangier disease, Tay-Sachs disease,Adrenoleukodystrophy, Type 2 Diabetes, Gaucher disease, Hereditaryhemochromatosis, Lesch-Nyhan syndrome, Maple syrup urine disease, Menkessyndrome, Niemann-Pick disease, Pancreatic cancer, Prader-Willisyndrome, Porphyria, Refsum disease, Tangier disease, Wilson's disease,Zellweger syndrome, progerias, SCID.

Autoimmune Disorders:

Autoimmune polyglandular syndrome, lupus, type I diabetes, scleroderma,multiple sclerosis, Crohn's disease, chronic active hepatitis,rheumatoid arthritis, Graves' disease, myasthenia gravis, myositis,antiphospholipid syndrome (APS), uveitis, polymyositis, Raynaud'sphenomenon, and demyelinating neuropathies, and rare disorders such aspolymyalgia rheumatica, temporal arteritis, Sjogren's syndrome, Bechet'sdisease, Churg-Strauss syndrome, and Takayasu's arteritis.

Inflammatory Disorders:

Alopecia, Diastrophic dysplasia, Ellis-van Creveld syndrome, Asthma,Arthritis, including osteoarthritis, rheumatoid arthritis, andspondyloarthropathies.

Age-Related Disorders:

Alzheimer Disease, Parkinson's Disease, Atherosclerosis, Age-RelatedMacular Degeneration, Age-related Osteoporosis.

The disclosed methods and compositions can also be used to treat,manage, or reduce symptoms associated with aging, in tissueregeneration/regenerative medicine, stem cell transplantation, inducingreversible genetic modifications, expressing inhibitory RNA, cognitiveenhancement, performance enhancement, and cosmetic alterations to humanor non-human animal.

D. Research Tools

In one embodiment, the present disclosure is used as a tool toinvestigate cellular consequences of gene expression. Mutant mice can begenerated using this approach, allowing investigators to study variousbiological processes. More particularly, the methods and compositionsdisclosed herein can be used to generate cells that contain unique genemodifications known in the art and at the discretion of one skilled inthe art.

E. Transgenic Non-Human Animals

The techniques described in the present disclosure can also be used togenerate transgenic non-human animals. In particular, zygotemicroinjection, nuclear transfer, blastomere electrofusion andblastocyst injection of embryonic stem (ES) cell cybrids have eachprovided feasible strategies for creating transgenic animals. In oneembodiment an embryonic stem (ES) cell is transfected and injected intothe blastocyst of a mammalian embryo as a means of generating chimericmice. In another embodiment, embryonic stem (ES) cell are firstprepared, followed by blastocyst injection into embryos. The use ofcells carrying specific genes and modifications of interest allows thecreation and study of the consequences of the transfected DNA. Intheory, this technique offers the prospect of transferring anypolynucleotide into a whole organism. For example, the disclosed methodsand compositions could be used to create mice possessing the deliveredpolynucleotide in a specific cell type or cell state.

Another embodiment of the disclosure provides transfected non-humanorganisms and methods making and using them. Single or multicellularnon-human organisms, preferably non-human mammals, more preferably mice,can be transfected with the compositions described herein byadministering the compositions of the present disclosure to thenon-human organism. In one embodiment, the polynucleotide remainsepisomal and does not stably integrate into the genome of the hostorganism. In another embodiment, the polynucleotide prevents theexpression of a gene of interest. Thus, the expression of thepolynucleotide in specific cells of the host can be controlled by theamount of polynucleotide administered to the host.

The disclosed transfected non-human organisms have several advantagesover traditional transgenic organisms. For example, the transfectedorganism disclosed herein can be produced in less time that traditionaltransgenic organisms without sexual reproduction. Moreover, theexpression of the polynucleotide of interest in the host can be directlyregulated by the amount of polynucleotide of interest administered tothe host. Dosage controlled expression of a polynucleotide of interestcan be correlated to observed phenotypes and changes in the transfectedanimal. Additionally, inducible expression and/or replication controlelements can be included in the polynucleotide of interest to provideinducible and dosage dependent expression and/or replication. Suitableinducible expression and/or replication control elements are known inthe art. Furthermore, the effect of genes and gene modifications inspecific cell types and states can be studied without affecting theentire cells of the animal.

F. PEG-Blocking Containing Polymers

The polymers described herein can be used for drug delivery, forexample, in the formation of particles, such as microparticles ornanoparticles, or micelles which can release one or more therapeutic,prophylactic, and/or diagnostic agents in a controlled release mannerover a desirable period of time.

Various pH-responsive micelle nanocarriers have been investigatedpreviously. Such micelles are often formed via self-assembly ofamphiphilic block copolymers and consist of a hydrophilic (e.g. PEG)outer shell and a hydrophobic inner core capable of response to mediumpH. Typically, upon changing the medium pH from neutral or slightlybasic to mildly acidic, the micelle cores undergo accelerateddegradation, become completely soluble in water, or swell substantiallyin aqueous medium. As the result, the drug-encapsulated micelles with aslow drug-release rate at the physiological pH can be triggered by anacidic pH to rapidly unload the drug molecules. The polymer segmentsconstituting the micelle cores in previous reports include poly(orthoesters), poly(β-amino esters), poly(L-histidine), and others. The majordisadvantages with most of the previous micelle systems are the multiplesteps required for preparing the copolymers and the difficulty ofcontrolling the polymer molecular weight and adjusting the polymercomposition during the copolymer synthesis.

The copolymers described herein exhibited variation in the rate ofrelease as a function of pH. In vitro drug release behaviors of theDTX-encapsulated micelles of PEG2K-PPMS copolymer samples(PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL) werestudied in PBS solution at both physiological pH of 7.4 and acidic pH of5.0. In general, the DTX release from all micelle samples followedbiphasic release kinetics and exhibited remarkable pH-dependence. TheDTX-loaded PEG2K-PPMS copolymer micelles release 25-45% drug rapidlyduring the initial 12 h, followed by a more gradual release ofadditional 25-40% drug for the subsequent 132 h. The influence of themedium pH on the drug release rate is substantial. For example, at theend of the incubation period (144 h), the values of accumulated DTXreleased from the micelles of PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL,and PEG2K-PPMS-51% PDL copolymers are respectively 66%, 60%, and 55% atphysiological pH of 7.4, which increase correspondingly to 85%, 81%, and75% at acidic pH of 5.0. The observed pH-triggered acceleration of DTXrelease from the PEG2K-PPMS copolymer micelles is consistent with theearlier observation that changing of the medium pH from 7.4 to 5.0causes significant swelling of the micelles due to the protonation andsize increase of the micelle PPMS cores. This pH-triggered micelle sizeexpansion would certainly facilitate the diffusion and release ofentrapped DTX from the micelle cores to the aqueous medium. On the otherhand, at a given pH, the DTX release rate is presumably controlled bythe interactions between the drug and the PPMS matrix in the micellecores. Since PDL-rich PEG2K-PPMS copolymers are expected to form stronghydrophobic domains in their micelle inner cores to better trap andretain hydrophobic DTX molecules, the drug release from such copolymermicelles should be more gradual and sustained. This hypothesis issupported by the experimental result showing that at both pH of 7.4 and5.0, the DTX release rate from PEG2K-PPMS copolymer micelles decreaseswith increasing PDL content in the PPMS chain segments of the copolymer.

It is known that upon uptake of micelles by tumor cells, the micelleparticles are subjected to entrapment in endosomes with pH ranging from5.5 to 6.0 and in lysosomes with pH ranging from 4.5 to 5.0. As theabove results clearly show, these acidic environments would inevitablytrigger fast DTX release from PEG2K-PPMS copolymer micelles, thusenhancing the cytotoxicity of the drug-loaded micelles. The amino groupsin the copolymers would act as proton sponges to facilitate endosomalescape. Therefore, the pH-responsive properties exhibited by thePEG2K-PPMS copolymer micelles are highly desirable, which render them tobe superior carriers for delivery of anticancer drugs.

The cytotoxicity of blank copolymer micelles, DTX-loaded copolymermicelles, and free DTX was evaluated on SK-BR-3 cells at pH of 7.4 usingthe MTT assay. The blank micelles exhibited no obvious cytotoxicity onSK-BR-3 cells as the cell viabilities of all treated cell groups wereover 90%. For example, after treatment with the micelles ofPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDLcopolymers at concentration as high as 200 μg/mL, the three treated cellgroups had respectively 97%, 94%, and 90% of the cells remaining viable.As expected, the cell viability decreases with increasing concentrationof DTX either in the form of the free drug or in the form of the drugencapsulated in the micelles. To quantify the in vitro efficacy of thesemicelle formulations, IC₅₀ values for DTX-loaded micelles of copolymersPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS −51% PDL werecalculated to be 0.57 nM, 0.83 nM, and 1.42 nM, respectively. Incomparison, the IC₅₀ value for free DTX was found to be 1.27 nM. IC₅₀ isdefined as the drug concentration of a specific formulation which isrequired to kill 50% of cells after they are incubated with the drug fora designated time period (48 h). Thus, except the PEG2K-PPMS-51% PDLcopolymer micelles, both DTX-loaded PEG2K-PPMS-11% PDL copolymermicelles and DTX-loaded PEG2K-PPMS-30% PDL copolymer micelles possesssignificantly higher cytotoxicity against SK-BR-3 cells than free DTX.The exceptionally high efficacy observed for DTX-loaded PEG2K-PPMS-11%PDL copolymer micelles is likely attributed to fast cellular uptake ofthe micelles and anticipated rapid intracellular DTX release from themicelles upon entrapment of the micelle particles in acidicendosomes/lysosomes.

The rate of DTX release from the micelles, particularly pH-triggeredacceleration of the drug release, appears to play a more important rolethan the cellular uptake in influencing the cytotoxicity of theDTX-loaded micelles. Thus, although the cellular uptake is faster forDTX-loaded PEG2K-PPMS-51% PDL copolymer micelles vs. DTX-loadedPEG2K-PPMS −30% PDL copolymer micelles (FIG. 8), the latter micellesrelease the drug at a higher rate to exert higher cytotoxicity towardSK-BR-3 cells.

The drug-loaded micelle particles were readily absorbed by SK-BR-3 cellsand were able to escape from entrapment by endosomes and lysosomes afterthe cellular uptake. Because of these desirable properties, DTX-loadedmicelles prepared from the copolymers described herein (e.g.,PEG2K-PPMS-11% PDL and PEG2K-PPMS-30% PDL) with low PDL content, highprotonation capability, and fast drug release at an acidic pH exertsubstantially higher potency against SK-BR-3 cancer cells than free DTXdrug. These results demonstrate that copolymer micelles have greatpotential to serve as pH-responsive nano-carriers for controlled releasedelivery of anticancer agents, such as DTX, to treat cancers.

G. Kits

Kits or packs that supply the elements necessary to conduct transfectionof eukaryotic or prokaryotic organisms, in particular the transfectionof specific cell types or cell states are also disclosed. In accordancewith one embodiment a kit is provided comprising the disclosed polymers,and optionally a polyplex coating, for example a target specificcoating. The polymer can be combined with a polynucleotide of the user'schoosing to form a complex which can be used to transfect a host or ahost cell. The polyplex can be further mixed with the coating to providecell-type or cell-state specific tropism.

The individual components of the kits can be packaged in a variety ofcontainers, e.g., vials, tubes, microtiter well plates, bottles, and thelike. Other reagents can be included in separate containers and providedwith the kit; e.g., positive control samples, negative control samples,buffers, cell culture media, etc. Preferably, the kits will also includeinstructions for use.

EXAMPLES Example 1 Synthesis and Characterization ofPoly(Amine-Co-Ester) Terpolymers

Materials and Methods

Materials

ε-Caprolactone (CL, 99%), 12-dodecanolide (DDL, 98%), ω-pentadecalactone(PDL, 98%), 16-hexadecanolide (HDL, 97%), diethyl sebacate (DES, 98%),N-methyldiethanolamine (MDEA, 99+%), N-phenyldiethanolamine (PDEA, 97%),and diphenyl ether (99%) were purchased from Aldrich Chemical Co. andwere used as received. Chloroform (HPLC grade), dichloromethane (99+%),hexane (97+%), methanol (98%), and chloroform-d were also obtained fromAldrich Chemical Co.

Immobilized Candida antarctica lipase B (CALB) supported on acrylicresin or Novozym 435 (Aldrich Chemical) dried at 50° C. under 2.0 mmHgfor 20 h prior to use.

Synthesis and Purification of Lactone-DES-MDEA Terpolymers

The copolymerization of a lactone with a diacid or diester (e.g.,diethyl sebacate (DES)), and a dialkyl amine (e.g.,N-methyldiethanolamine (MDEA)) was performed in diphenyl ether solutionusing a parallel synthesizer connected to a vacuum line with the vacuum(±0.2 mmHg) controlled by a digital vacuum regulator. In a typicalexperiment, reaction mixtures were prepared, which contained threemonomers (lactone, DES, and MDEA), Novozym 435 catalyst (10 wt % vs.total monomer), and diphenyl ether solvent (200 wt % vs. total monomer).The copolymerization reactions were carried out at a constanttemperature in two stages: first stage oligomerization, followed bysecond stage polymerization. The reaction temperature was 80° C. for thereactions of ε-caprolactone (CL) with DES and MDEA and was set at 90° C.for the copolymerizations of all other lactones [12-dodecanolide (DDL),15-pentadecanolide (PDL), and 16-hexadecanolide (HDL)] with DES andMDEA.

During the first stage reaction, the reaction mixtures were stirredunder 1 atm of nitrogen gas, after which the reaction pressure werereduced to 1.6 mmHg and the reactions were continued for additional 72h. The terpolymer products were isolated and purified according to thefollowing procedures.

Because the solubility and physical properties of the terpolymers varysubstantially depending on the ring size of the lactones and the lactoneunit content in the polymers, two different purification methods weredeveloped to isolate the polymer products. For purification of thosepolymers that are viscous liquids or waxy solids (e.g., CL-DES-MDEAterpolymers with ≦80% CL, DDL-DES-MDEA terpolymers with ≦40% DDL,PDL-DES-MDEA terpolymers with ≦20% PDL, and HDL-DES-MDEA terpolymerswith ≦20% HDL), the crude product mixtures were first mixed with hexaneto cause the precipitation of the polymers. The precipitated polymerswere then washed several times with fresh hexane to extract and removethe residual diphenyl ether solvent from the polymers. Subsequently, theterpolymers were dissolved in dichloromethane and filtered to removecatalyst particles. Evaporation and complete removal of the CH₂Cl₂solvent from the filtrates at 40° C. under high vacuum (1.0 mmHg)yielded the purified terpolymers.

For purification of the solid lactone-DES-MDEA terpolymers (e.g.,DDL-DES-MDEA terpolymers with >40% DDL, PDL-DES-MDEA terpolymerswith >20% PDL, and HDL-DES-MDEA terpolymers with >20% HDL), the crudeproduct mixtures were first dissolved in chloroform. The resultantpolymer solutions were then filtered to remove the enzyme catalyst.After being concentrated under vacuum, the filtrates were added dropwiseto stirring methanol to cause precipitation of the terpolymers. Theobtained white solid polymers were subsequently washed with methanolthree times and dried at 40° C. under high vacuum (1.0 mmHg) for 16 h.The isolated yield, composition, molecular weight (M_(w)), andpolydispersity (M_(w)/M_(n)) of the synthesized terpolymers are reportedin Table 1.

Size Determination of Polyplexes

Size of the polyplexes was determined by Zetasizer (Malvern). Thepolyplex nanoparticles were placed on a round cover glass mounted on analuminum stub using carbon adhesive tape. After drying at roomtemperature, the stub was sputter-coated with a mixture of gold andpalladium (60:40) under low pressure of argon using a Dynavac MiniCoater and subjected to SEM analysis.

Determination of Amount of III-20% PDL Polymer Associated with DNA

Size exclusion chromatography was used to determine the amount ofIII-20% PDL polymer associated with or without DNA when mixed with DNAat a 100:1 weight ratio. Size exclusion chromatography was performedusing a Sepharose CL-2B column (14.5×50 mm, 8.3 ml column volume) at aflow rate of 0.4 ml/min. Twenty percentage of N-methyldiethyleneamine inIII-20% PDL polymer was replaced with N-phenyldiethyleneamine, whichallows sensitive detection of polymer based on UV absorption. PlasmidDNA was labeled with Cy3 Label IT® Tracker™ Intracellular Nucleic AcidLocalization Kit (Mirus Bio LLC) following manufacturer's protocol.Columns were pre-conditioned with 100 μg free III-20% PDL to preventnon-specific interactions between the polymer and column. After columnequilibriation, fractions (0.1 ml) were collected using a phosphatebuffered saline (pH 7.4) elution buffer. Elution samples were analyzedby absorbance at 300 nm and spectrofluorescence (ex/em: 550/570). Theresults are illustrated in FIG. 6. Elution fractions were monitored forboth III-20% PDL polymer (•) and Cy3-DNA (□) content after loadingIII-20% PDL/Cy3-DNA polyplexes on a Sepharose CL-2B column. FIG. 6 showsrepresentative data from three separate experiments. The amount ofDNA-associated polymer was determined by area under curve analysis.

Instrumental Methods

¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE 500spectrometer. The chemical shifts reported were referenced to internaltetramethylsilane (0.00 ppm) or to the solvent resonance at theappropriate frequency.

The number and weight average molecular weights (M_(n) and M_(w),respectively) of polymers were measured by gel permeation chromatography(GPC) using a Waters HPLC system equipped with a model 1515 isocraticpump, a 717 plus autosampler, and a 2414 refractive index (RI) detectorwith Waters Styragel columns HT6E and HT2 in series. Empower II GPCsoftware was used for running the GPC instrument and for calculations.Both the Styragel columns and the RI detector were heated and maintainedat 40° C. temperature during sample analysis. Chloroform was used as theeluent at a flow rate of 1.0 mL/min. Sample concentrations of 2 mg/mLand injection volumes of 100 μL were used. Polymer molecular weightswere determined based on a conventional calibration curve generated bynarrow polydispersity polystyrene standards from Aldrich Chemical Co.

Results

PMSC was shown previously to be an efficient vector for in vitrotransfection of cell lines and for direct intratumoral gene delivery invivo, but was not able to deliver genes after systemic administration.To determine if PMSC chain structures with additional hydrophobic repeatunits might lead to more efficient gene vectors a new method for thesynthesis of terpolymers from lactone, diethyl sebacate (DES), andN-methyldiethanolamine (MDEA) was developed using Candida antarcticalipase B (CALB) as catalyst. CALB is an efficient catalyst for combinedring-opening and condensation copolymerization of lactone with dialkyldiester and conventional diol monomers.

The terpolymerization of DES and MDEA with various ring size lactoneswas performed in two stages: oligomerization under 1 atmosphericpressure of nitrogen, followed by polymerization at 1.6 mmHg vacuum(FIG. 1). During the initial oligomerization, the monomers wereconverted to nonvolatile oligomers. The subsequent use of high vacuumfacilitates removal of the byproduct ethanol, thus accelerating polymerchain growth. This method allowed for the synthesis of novelpoly(amine-co-esters) with diverse chain structures and tunablehydrophobicity.

Using this method, lactones in a wide range of ring sizes (C₄ to C₂₄,preferably C₆ to C₁₆) can serve as comonomers and the copolymerizationreactions were accomplished in one step without protection anddeprotection of the amino group of MDEA. Such amino-bearing copolyesterswould be extremely difficult to synthesize using conventionalorganometallic catalysts since metal catalysts are often sensitive to(or deactivated by) organic amines and are known to be inefficient forpolymerizing large ring lactone monomers (Stridsberg, et al., Controlledring-opening polymerization: Polymers with designed macromoleculararchitecture. Degradable Aliphatic Polyesters 157, 41-65 (2002))(Nomura, et al. Anionic Ring-Opening Polymerization of MacrocyclicEsters. Macromolecules 27, 620-621 (1994)).

Furthermore, enzymatic polymerization catalysis has distinct advantagesfor producing biomedical polymers due to the high activity andextraordinary selectivity of enzyme catalysts and resultant high purityof products that are also metal-free (DeRouchey, et al. Structuralinvestigations of DNA-polycation complexes. Eur Phys J E Soft Matter 16,17-28 (2005)). In design of the current polycationic gene carriers,lactone was chosen as one of the comonomers because the hydrophobicityof lactone-DES-MDEA terpolymers could be effectively altered by choosinga lactone with a specific ring-size and/or by adjusting lactone unitcontent in the terpolymers. In addition, readily available lactones ofvarious ring size are known to possess low toxicity: for example,polyesters derived from small lactones, such as poly(ε-caprolactone) andpoly(p-dioxanone), are commercial biomaterials and have been used inclinical applications. Large (e.g., C₁₆-C₂₄) lactones and theirpolyester derivatives are natural products that were found to be presentin several different types of bees.

Table 1, above, shows the yield, composition, molecular weight,polydispersity, and other characterization data of selectedlactone-DES-MDEA terpolymers that were prepared as described above.Table 1 includes data from samples that have low solubility in polarorganic solvents (e.g., DMSO) and thus were not able to form polyplexeswith DNA. To simplify nomenclature, CL-DES-MDEA, DDL-DES-MDEA,PDL-DES-MDEA, and HDL-DES-MDEA terpolymers are designated as polymer I,II, III, and IV, respectively (Table 1A). The composition of eachindividual terpolymer is further denoted as x % lactone indicating thelactone unit content [mol % vs. (lactone+sebacate) units] in thepolymer. For example, II-40% DDL and III-20% PDL represent DDL-DES-MDEAcopolymer with 40% DDL and PDL-DES-MDEA copolymer with 20% PDL,correspondingly.

The lactone-DES-MDEA terpolymers were obtained in good yields (80-86%)and the composition of the terpolymers were readily controlled byadjusting the corresponding monomer feed ratio (Table 1). The molecularweight (M_(w)) of the polymers ranged from 18000 to 39000 withpolydispersity (M_(w)/M_(n)) between 1.8 and 2.3. Compared to PEI, whichcontains 32.6 wt % nitrogen, the lactone-DES-MDEA terpolymers had lownitrogen contents (1.9-4.7 wt %). In general, the solubility oflactone-DES-MDEA terpolymer in DMSO decreases with increasing lactonering size at a given lactone content. Among terpolymers synthesized froma same lactone, solubility in DMSO is lower at a higher lactone content.

The lactone-DES-MDEA terpolymers were characterized by ¹H and ¹³C NMRspectroscopy. The polymer chains consist of three different types ofrepeat units: lactone, dialkyl amine (e.g., N-methyldiethyelenamine(MDEA)), and a diester (e.g., sebacate) (FIG. 1). Proton NMR spectrawere used to measure the composition (repeat unit ratio) of theterpolymers. The repeat unit sequence distributions (diad distributions)in the polymers were analyzed by ¹³C NMR spectroscopy and theexperimental results were compared to the values calculated forstatistically random terpolymers at same compositions. Consistent withthe microstructures of PDL-diethyl succinate-1,4-butanediol terpolymersthat were prepared previously using the same catalyst, the unitarrangements in lactone-DES-MDEA copolymers were also random. Thus,these polymers can also be described aspoly(lactone-co-N-methyldiethyleneamine-co-sebacate).

Because of branching nature of the tertiary amino groups in polymerchains, PMSC was a viscous liquid at ambient temperature. Incorporationof lactone into the poly(amine-co-ester) resulted in lactone-DES-MDEAterpolymers in which the physical properties vary substantiallydepending on the ring size of the lactone and its content in thepolymers. In general, the terpolymers with a small ring lactone and lowlactone content are liquids and those with a large lactone and a highlactone content are waxy or solid materials. Thus, I-(10-80)% CL,II-(10-40)% DDL, III-(10-20)% PDL, and IV-10% HDL were viscous liquidsat room temperature while II-(60-80)% DDL, III-(40-80)% PDL, andIV-(20-80)% HDL were either semisolid or solid polymers (Table 1).

Synthesis and Purification of Lactone-DES-MDEA

Polymers

General procedures for CALB-catalyzed terpolymerization of lactone withDES and MDEA and the procedures for isolation and purification of theformed terpolymer products are described above. The ¹H and ¹³C NMRresonance absorptions of the polymers are shown below. Table 1summarizes the yield, composition, molecular weight (M_(w)),polydispersity (M_(w)/M_(n)), and nitrogen content of all purifiedLactone-DES-MDEA terpolymers.

CL-DES-MDEA terpolymer (I): ¹H NMR (CDCl₃; ppm) 1.29 (br.), 1.34-1.39(m), 1.60 (br.), 2.25-2.30 (m), 2.32 (s), 2.67 (t), 4.04 (t), 4.15 (t);¹³C NMR (CDCl₃; ppm) 24.48, 24.53, 24.84, 24.89, 25.46, 25.48, 28.32,29.02-29.06 (m), 33.97, 34.02, 34.15, 34.20, 42.83, 55.91, 61.91, 61.97,63.92, 64.01, 173.21, 173.31, 173.50, 173.60, plus a small absorption at14.22 ppm.

DDL-DES-MDEA terpolymer (II): ¹H NMR (CDCl₃; ppm) 1.27-1.29 (br.), 1.61(m, br.), 2.26-2.31 (m), 2.34 (s), 2.69 (t), 4.05 (t), 4.16 (t); NMR(CDCl₃; ppm) 24.87, 24.91, 24.95, 25.00, 25.93, 28.66, 29.06, 29.08,29.14, 29.25, 29.27, 29.43, 29.50, 34.21, 34.24, 34.31, 34.35, 42.88,55.95, 61.95, 64.35, 173.63, 173.68, 173.81, 173.86, plus a smallabsorption at 14.25 ppm.

PDL-DES-MDEA terpolymer (III): ¹H NMR (CDCl₃; ppm) 1.26-1.29 (br.), 1.61(m, br.), 2.26-2.32 (m), 2.34 (s), 2.69 (t), 4.05 (t), 4.16 (t), plus asmall absorption (triplet) at 3.57 ppm due to —CH₂CH₂OH end groups; ¹³CNMR (CDCl₃; ppm) 24.84, 24.90, 24.92, 24.99, 25.92, 28.65, 29.03, 29.07,29.12, 29.25, 29.28, 29.47, 29.53, 29.58-29.63 (m), 34.13, 34.18, 34.23,34.28, 42.77, 55.87, 61.86, 64.27, 173.44, 173.50, 173.62, 173.68, plustwo small absorptions at 14.22 and 61.58 ppm due to —CO—OCH₂CH₃ terminalgroups.

HDL-DES-MDEA terpolymer (IV): ¹H NMR (CDCl₃; ppm) 1.26-1.29 (br.), 1.60(m, br.), 2.25-2.31 (m), 2.32 (s), 2.68 (t), 4.05 (t), 4.15 (t); ¹³C NMR(CDCl₃; ppm) 24.86, 24.91, 24.94, 25.00, 25.92, 28.65, 29.05, 29.07,29.14, 29.25, 29.28, 29.47, 29.53, 29.59-29.65 (m), 34.18, 34.23, 34.28,34.34, 42.86, 55.94, 61.94, 64.33, 173.57, 173.64, 173.75, 173.82, plusa small absorption at 14.24 ppm.

Synthesis of Purified PDL-DES-MDEA-PDEA Copolymer

A reaction mixture containing PDL/DES/MDEA/PDEA monomers in a molarratio of 1:9:7.2:1.8, Novozym 435 catalyst (10 wt % vs. total monomer),and diphenyl ether (200 wt % vs. total monomer) was stirred at 90° C.under 1 atmosphere of nitrogen for 19 h. Subsequently, the reactionpressure was reduced to 1.2 mmHg and the reaction was continued at 90°C. for additional 72 h. The resultant, liquid PDL-DES-MDEA-PDEAcopolymer was purified according to a procedure similar to that used forpurification of PDL-DES-MDEA terpolymer III-10% PDL as described above.This copolymer consists of four repeat units: PDL, sebacate,N-methyldiethyleneamine, N-phenyldiethyleneamine; and its compositionwas determined by ¹H NMR spectroscopy.

PDL-DES-MDEA-PDEA copolymer: yield=87%; 10 mol % PDL vs. (PDL+sebacate);20 mol % PDEA vs. (MDEA+PDEA); M_(w)=243000; M_(w)/M_(n)=1.8; ¹H NMR(CDCl₃; ppm): 1.26-1.29 (br.), 1.61 (m, br.), 2.25-2.31 (m), 2.33 (s),2.68 (t), 3.58 (t, —CH₂—CH₂—N(Ph)-CH₂—CH₂—), 4.05 (t), 4.15 (t), 4.22(t, —CH₂—CH₂—N(Ph)-CH₂—CH₂—), 6.69 (t), 6.75 (d), 7.20 (t).

Study on Polymer Chain Growth During Lactone-DES-MDEA

Terpolymerization

Terpolymerization of PDL with DES and MDEA was selected as a typicalexample for polymer chain growth study. The reaction mixture contained2:3:3 (molar ratio) PDL/DES/MDEA comonomers, Novozym 435 catalyst (10 wt% vs. total monomer), and diphenyl ether solvent (200 wt % vs. totalmonomer). The copolymerization reactions were carried out at 60, 70, 80,and 90° C. temperatures in two stages: first stage oligomerization under1 atmosphere pressure of nitrogen for 19 h, followed by second stagepolymerization under 1.4 mm Hg vacuum for 72 h. To monitor the polymerchain growth, aliquots were withdrawn at various time intervals duringthe second stage polymerization. The formed polymers were then dissolvedin HPLC-grade chloroform and filtered to remove the enzyme catalyst.Polymer products were not fractionated by precipitation prior toanalysis of molecular weight. The filtrates containing whole productswere analyzed by GPC using narrow polydispersity polystyrene standardsto measure polymer molecular weights.

FIG. 2 shows the changes in polymer molecular weight (M_(w)) vs.polymerization time for the copolymerization at different temperatures.For all reactions, continuous chain growth was observed during the 72hour polymerization period. For example, at 4, 21, 31, 47, 55, 72 h, theproducts formed at 90° C. had M_(w) values of 12700, 19300, 21100,26100, 30200, and 39700, respectively. Among these reactions, thecopolymer molecular weight at a given reaction time was found toincrease with increasing reaction temperature from 60 to 90° C. Thus, at72 h, the resultant copolymers of the reactions at 60, 70, 80, and 90°C. possessed M_(w) values of 13300, 19200, 32300, and 39700,correspondingly. These results indicate that the molecular weight of thePDL-DES-MDEA terpolymers could be readily controlled by adjusting thereaction time and/or reaction temperature. The product polydispersity(M_(w)/M_(n)) vs. molecular weight (M_(w)) for the copolymerizationreactions is delineated in FIG. 2B. The polydispersity values of allproducts follow a similar trend, which changed from 1.5 to 1.7 withincreasing polymer molecular weight (M_(w)) from 6800 to 19000, andremained fairly constant at 1.8 in the molecular weight range between19000 and 40000. Furthermore, NMR analysis showed that during thecopolymerization reactions, byproduct ethanol was formed and condensedin the dry ice trap between the reactors and vacuum pump.

To determine whether the polymerization reactions were indeed catalyzedby CALB, control experiments were performed without the lipase. Thecontrol reaction was carried out at 90° C. in diphenyl ether underidentical conditions (stage 1: 2:3:3 PDL/DES/MDEA monomer ratio, under 1atmosphere pressure of nitrogen for 19 h; stage 2: 1.4 mmHg for 72 h).GPC analysis showed that the product had a M_(w) of less than 800. Thisdemonstrates that CALB catalyzes lactone-DES-MDEA terpolymerization.

Structural Characterization of Lactone-DES-MDEA

Terpolymers

The lactone-DES-MDEA terpolymers were characterized by ¹H and ¹³C NMRspectroscopy. The polymer chains consist of three different types ofrepeat units: lactone, N-methyldiethanolamine (MDEA), and sebacate (FIG.1). Proton NMR spectra were used to measure the composition (repeat unitratio) of the terpolymers. The repeat unit sequence distributions (diaddistributions) in the polymers were analyzed by ¹³C NMR spectroscopy andthe experimental results were compared to the values calculated forstatistically random terpolymers at same compositions. Consistent withthe microstructures of PDL-diethyl succinate-1,4-butanediol terpolymersthat were prepared previously using the same catalyst, the unitarrangements in lactone-DES-MDEA copolymers were also random (Jiang, Z.Lipase-catalyzed synthesis of aliphatic polyesters via copolymerizationof lactone, dialkyl diester, and diol. Biomacromolecules 9, 3246-3251(2008)) (Mazzocchetti, et al. Enzymatic Synthesis and Structural andThermal Properties ofPoly(omega-pentadecalactone-co-butylene-co-succinate). Macromolecules42, 7811-7819 (2009)). Thus, these polymers can also be described aspoly(lactone-co-N-methyldiethyleneamine-co-sebacate).

The structure and composition of the lactone-DES-MDEA terpolymers weredetermined by ¹H and ¹³C NMR spectroscopy. NMR resonance absorptionswere assigned by comparing signals of the terpolymers to those ofreference polymers, poly(lactone) homopolymers andpoly(N-methyldiethyleneamine sebacate) (PMSC), and by observing changesin signal intensities among the terpolymers synthesized from variouslactone/DES/MDEA monomer feed ratios.

The proton NMR spectra of the lactone-DES-MDEA terpolymers showed thatthe copolymers contained three different types of repeating units:lactone, N-methyldiethyleneamine, and sebacate. The molar ratios oflactone to N-methyldiethyleneamine to sebacate units in the terpolymerswere calculated from proton resonance absorptions: number of lactoneunits from methylene absorption at 4.05 (±0.01) ppm, number ofN-methyldiethyleneamine units from absorptions at 4.15 (±0.01) or 2.68(±0.01) ppm, and number of sebacate units from absorption at 1.60(±0.01) ppm after subtracting contribution from lactone units.

The above structural assignments for the terpolymers I-IV were furthersupported by the ¹³C NMR spectra of the polymers. All terpolymersexhibited four ester carbonyl resonance absorptions at 173.2-173.9 ppmdue to two diads of lactone unit and two diads of sebacate unit. Forterpolymers II, III, and IV that contain large (≧C₁₂) lactone units, thefour resonance peaks with decreasing chemical shift are attributable tolactone*-lactone, sebacate*-lactone, lactone*-MDEA, and sebacate*-MDEAdiads, respectively. For CL-DES-MDEA terpolymer (or terpolymer I), thecarbon-13 absorbances at 173.60, 173.50, 173.31, and 173.21 ppm areascribable to CL*-MDEA, sebacate*-MDEA, CL*-CL, and sebacate*-CL diads,correspondingly. Furthermore, for terpolymers II, III, and IV, the—CH₂O— group of the lactone units and the —CH₂O— group of the MDEA unitsresonated at 64.3 (±0.05) and 61.9 (±0.05) ppm, respectively.

It was found that increasing lactone content in the polymers resulted inhigher resonance intensity at 64.3 ppm, but lower absorbance intensityat 61.9 ppm. However, terpolymer I showed four resonance absorptions ofthe —CH₂O— groups at 64.01, 63.92, 61.97, and 61.91 ppm, which areattributable to CL*-CL, CL*-sebacate, MDEA*-CL, and MDEA*-sebacatediads, correspondingly. In support of this structural assignment, it wasobserved that increasing CL content in terpolymer I increases theabsorption intensity at 64.01 ppm, but decreases the absorptionintensity at 61.91 ppm. On the other hand, the intensities of the tworesonances at 63.92 and 61.97 ppm were comparable regardless of thepolymer composition. For all four terpolymers, the resonance absorptionsof the methyl and methylene groups adjacent to the nitrogen in MDEAunits appeared at 42.8 (±0.08) and 55.9 (±0.05) ppm, respectively.

To determine the repeat unit sequence distributions in the terpolymers,the abundance of lactone*-lactone (L*-L), lactone-MDEA (L*-M),sebacate*-lactone (S*-L), and sebacate*-MDEA (S*-M) diads in thepolymers was measured by ¹³C NMR spectroscopy and the obtainedexperimental values were then compared to theoretical diad distributionvalues calculated for random copolymers with same compositions. For acompletely random lactone-DES-MDEA terpolymer, distributions of L*-L,L*-M, S*-L, and S*-M diads can be calculated by the following equations:L*-L distribution=f _(L) ×f _(L)L*-M distribution=f _(L)×(2f _(M))S*-L distribution=(2f _(S))×f _(L)S*-M distribution=(2f _(S))×(2f _(M))where f_(L), f_(S), f_(M) are correspondingly molar fractions of L, S,and M repeating units in the terpolymers. It needs to be noted that inthe above formulae, molar fractions of S and M units are doubled becauseboth ends of the units can form an ester linkage of a diad, while onlyone end of L units can serve the purpose. Table 2 summarizes themeasured diad distributions of terpolymers I, II, III, and IV withdifferent compositions, as well as the values calculated for randomcopolymers. For all copolymers studied, the experimental values matchremarkably well with the calculated values. Thus, the lactone-DES-MDEAterpolymers synthesized using CALB catalyst contain lactone, sebacate,and N-methyldiethyleneamine repeat units randomly distributed in thepolymer chains, and can be described aspoly(lactone-co-N-methyldiethylenamine-co-sebacate). These results areconsistent with previous reports showing that enzymatic PDL-diethylsuccinate-1,4-butanediol terpolymers also possessed random chainstructures (Jiang, Z. Lipase-catalyzed synthesis of aliphatic polyestersvia copolymerization of lactone, dialkyl diester, and diol.Biomacromolecules 9, 3246-3251 (2008)).

TABLE 2 Diad Distributions of Lactone-DES-MDEA Terpolymers: Comparisonbetween Experimental Values and Theoretical Values Calculated for RandomCopolymers L/S/M^(b) L*-L L*-M S*-L S*-M Polymer^(a) (unit ratio)meas^(c) calc^(d) meas^(c) calc^(d) meas^(c) calc^(d) meas^(c) calc^(d)I-20% CL 20:80:80 0.01 0.01 0.10 0.10 0.10 0.10 0.80 0.79 I-40% CL40:60:60 0.06 0.06 0.19 0.19 0.19 0.19 0.56 0.56 I-60% CL 60:40:40 0.180.18 0.25 0.25 0.25 0.25 0.33 0.33 I-80% CL 80:20:20 0.45 0.45 0.22 0.220.22 0.22 0.11 0.11 II-20% DDL 20:80:80 0.02 0.01 0.11 0.10 0.11 0.100.76 0.79 II-40% DDL 40:60:60 0.06 0.06 0.20 0.19 0.20 0.19 0.54 0.56II-60% DDL 60:40:40 0.18 0.18 0.25 0.25 0.25 0.25 0.32 0.33 II-80% DDL80:20:20 0.46 0.45 0.22 0.22 0.22 0.22 0.10 0.11 III-40% PDL 40:60:600.06 0.06 0.18 0.19 0.20 0.19 0.56 0.56 III-82% PDL 82:18:18 0.48 0.480.22 0.21 0.20 0.21 0.10 0.09 IV-20% HDL 20:80:80 0.01 0.01 0.10 0.100.11 0.10 0.78 0.79 IV-40% HDL 40:60:60 0.06 0.06 0.19 0.19 0.20 0.190.55 0.56 IV 61% HDL 61:39:39 0.18 0.19 0.25 0.25 0.25 0.25 0.32 0.32IV-80% HDL 80:20:20 0.45 0.44 0.23 0.22 0.23 0.22 0.09 0.11 ^(a)SeeTable 1 for meanings of the polymer abbreviations. ^(b)Unitabbreviations: L for lactone; S for sebacate; M forN-methyldiethyleneamine. ^(c)Measured from ¹³C NMR spectra.^(d)Calculated for a copolymer with statistically random unitdistribution in the polymer chains.

Example 2 Terpolymers are Effective for Gene Delivery In Vitro

Materials and Methods

In Vitro Transfection and Characterization

Human embryonic kidney 293 (HEK293) cell line and lung cancer cell lineA549 were obtained from ATCC (American Type Culture Collection). Cellswere grown in DMEM medium (Invitrogen) supplemented with 10% fetalbovine serum (Invitrogen), 100 units/ml penicillin, and 100 μg/mlstreptomycin (Invitrogen) in a 37° C. incubator containing 5% CO₂. Forin vitro transfection, DNA polyplexes with weight ratio of 100:1 wereused unless otherwise noted. Polymers were dissolved in DMSO at 25mg/ml.

For preparing DNA polyplexes for transfection in 24 well plates, 4 μl ofpolymer solution (25 mg/ml in DMSO) was first diluted in 50 μl sodiumacetate buffer (25 mM, pH=5.2). After brief vortexing, the polymersolution was mixed with the same volume of a DNA solution containing 1μg DNA and vortexed for additional 10 seconds. The polymer/DNA mixturewas incubated at room temperature for 10 min and then added to cells,which were seeded in 24-well plates at density of 75,000 cells/well in500 μl of medium one night before transfection. Transfection usingLipofectamine 2000 (Invitrogen Corp.) was performed using the proceduresprovided by the manufacturer. PEI transfection was performed using thestandard protocol by keeping the weight ratio of PEI to DNA at 3. Thesame amount of DNA was used in experiments comparing lactone-DES-MDEAterpolymer with Lipofectamine 2000 and PEI.

For luciferase gene transfection, plasmid DNA expression luciferase,pGL4.13 (Promega) was used. Two days after transfection, the culturemedium was removed and the cells were washed with cold PBS. Two hundredmicro-liter Report Lysis Buffer (Promega) was added to each well. With afreeze-thaw cycle, cell lysate was collected. After a quick spin, 20 μlwas subjected to luciferase assay using Luciferase Assay Reagentaccording to the standard protocol described in manufacturer manual(Promega). Additional 25 μl was used to quantify protein content usingPierce BCA protein assay kit (Pierce, Thermo Scientific). Luciferasesignal was divided by the amount of total protein for comparison.Internal controls were used for normalization for group by groupcomparison. For experiments to detect cytotoxicity due to TRAIL, plasmidpEGFP-TRAIL (Kagawa, et al. Antitumor activity and bystander effects ofthe tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene. Cancer Res 61, 3330-3338 (2001)) (Addgene) was used. Controlplasmid, pEGFP, was obtained by removing TRAIL gene from pEGFP-TRAIL.Cell proliferation was determined by a standard MTT assay five daysafter transfection. The particle size and zeta potential of freshlyprepared polyplexes were measured by ZetaPals dynamic light scattering(Brookhaven Instruments Corp). The morphology of polyplexes, which wasstained with uranyl acetate, was visualized using FEI Tencai Biotwin TEMat 80 Kv. Images were taken using Morada CCD and iTEM (Olympus)software.

Results

All liquid terpolymers were evaluated for luciferase gene transfectionon HEK293 and A549 cells. Gene transfection efficiency increased withincreasing lactone content for both terpolymer II and III (FIG. 3A).Thus, II-40% DDL transfected A549 cells with an efficiency that is 2,162, and 1047 times higher than that of II-20% DDL, II-10% DDL, andPMSC, respectively. On the other hand, III-20% PDL was 6 and 1259 timemore efficient than III-10% PDL and PMSC, respectively, in transfectingA549 cells (FIG. 3A). A similar trend was observed for transfectingHEK293 cells (FIG. 3A). Although group I terpolymers in general weremore effective gene carriers than PMSC, the correlation between theirtransfection efficiency and lactone (CL) content does not follow asimple, consistent trend as observed for terpolymers II and III. Lactonering size also significantly affects the gene delivery performance ofthe terpolymers. At a given lactone content, terpolymers with long chainlactone units deliver genes with higher efficiency than those with shortchain lactone units. For example, the efficiency of HEK293 celltransfection was 27 times higher for III-10% PDL vs. II-10% DDL, and was27 times higher for III-20% PDL vs. II-20% DDL (FIG. 3A). Similarlactone size effects were observed for the transfection of A549 cells(FIG. 3A). These results demonstrate that gene transfection efficiencyof lactone-DES-MDEA terpolymers can be improved by using a large lactoneand by adjusting lactone content in the polymers. The remarkable effectsof lactone ring size and lactone unit content on gene transfectionperformance of lactone-DES-MDEA terpolymers support our hypothesis thathydrophobicity plays an important role in influencing transfectionefficiency of cationic polymers.

Among all terpolymers evaluated, III-20% PDL showed the best genedelivery. Terpolymer III with a higher PDL content (e.g., III-40% PDLand III-60% PDL) and lactone-DES-MDEA terpolymers with a larger lactone(e.g., group IV terpolymers) had low solubility in polar organicsolvents (e.g., DMSO), and were not able to form polyplexes in aqueoussolution. Despite the fact that III-20% PDL has lower nitrogen densitythan PMSC (Table 3), the optimal polyplex composition for gene deliverywas the same (at 100:1 weight ratio of polymer to DNA) for both polymers(FIG. 3B) (Kafil, et al. Cytotoxic Impacts of Linear and BranchedPolyethylenimine Nanostructures in A431 Cells. BioImpacts 1, 23-30(2011)).

TABLE 3 Physical properties of polyplex nanoparticles formed from DNA(pGL4.13) and lactone-DES-MDEA terpolymer N/P** Mean particle Zetapotential Polymer name* (molar ratio) radius (nm) (mV) PMSC*** 116 7015.7 I-10% CL 111 73 11.3 I-20% CL 105 107 10.8 I-40% CL 91 45 15.3I-60% CL 72 43 16.7 I-80% CL 45 117 7.3 II-10% DDL 107 69 10.4 II-20%DDL 99 93 11.6 II-40% DDL 79 90 8.9 III-10% PDL 106 86 13.2 III-20% PDL96 75 8.9 IV-10% HDL 105 — — *See Table 1 for polymer nomenclature.**All polyplex nanoparticles were formed at 100:1 weight ratio(polymer/DNA). ***PMSC is included here as a reference polymer.

There was a dramatic increase in transfection efficiency betweenparticles with 25:1 and 50:1 polymer to DNA ratio, which potentially canbe explained by the difference in size of these complexes (FIG. 4). Apolymer to DNA ratio of 100:1 was selected for all subsequentexperiments: at this ratio, nanoparticle complexes are spherical inshape as determined by both TEM and SEM. III-20% PDL/DNA complexes aremore stable than traditional polyplexes, such as PEI/DNA complexes:incubation of complexes in 2% heparin released 65% of DNA from III-20%PDL/DNA complexes, compared to 97% for PEI/DNA complexes (FIG. 5). In atypical III-20% PDL/DNA suspension, 80% of III-20% PDL is associatedwith DNA (FIG. 6). Due to the low charge density and predominantlyhydrophobic composition of III-20% PDL, the terpolymer that is notassociated with DNA is water insoluble, so the rest of the 20% ofterpolymer is likely present as particulates (FIG. 6). Furtherexperiments demonstrated that III-20% PDL with 100:1 polymer to DNAratio was sufficient to condense DNA (FIG. 7) and protect DNA fromenzymatic degradation (FIG. 8).

At the 100:1 weight ratio, III-20% PDL transfected A549 cells with 81and 166 times higher efficiency than Lipofectamine 2000 and PEI,respectively. For transfection of HEK293 cells, III-20% PDL was ˜50times more efficient than Lipofectamine 2000 and PEI (FIG. 3A). However,under conditions that allow optimal transfection efficiency,Lipofectamine 2000 and PEI are toxic. Incubation of complexes of DNAwith Lipofectamine 2000 at the optimal transfection conditions in thisstudy inhibited cell proliferation by over 50%. PEI has similartoxicity. For this reason, Lipofectamine and PEI are normally used totransfect cells only at high confluence, where sufficient numbers ofcells can survive. III-20% PDL is much less toxic than Lipofectamine andPEI (FIGS. 3C and 3D). PEI killed all cells three days after treatmentat 10 μg/ml. In contrast, III-20% PDL was non-toxic even atconcentrations as high as 500 μg/ml (FIGS. 3C and 3D).

Example 3 Terpolymers are Effective for Gene Delivery In Vivo

Materials and Methods

Preparation of Coated Polyplexes for In Vivo Evaluations

To prepare polyplexes for in vivo gene delivery, 40 μl polymer solutionin DMSO (50 mg/ml) was diluted to 40 μl NaAc buffer. After briefvortexing, the polymer solution was mixed with 80 μl NaAc buffercontaining 0.25 mg/ml plasmid DNA, followed by a vigorous vortex for 10second. Coating polyplexes with peptide polyE-mRGD was conducted 10 minafter incubation at room temperature, by adding 40 μl buffer containingpeptide at 2.5 mg/ml and allowing further incubation for 5 min. PeptidepolyE-mRGD (EEEEEEEEEEEEEEEE-GGGGGG-RGDK (SEQ ID NO:1)) was synthesizedat the W.M. Keck Facility at Yale University. Immediately beforeinjection, another 40 μl buffer containing 30% glucose was added. Twohundred microliter of the resulted mixture was then injected throughtail vein of each mouse.

To test the effect of serum of polyplex surface change, polyplexes wereprepared by mixing polymer and DNA and incubating at room temperaturefor 10 min. Then, polyE-mRGD was added at various concentrations andcoating allowed for 5 min. The zeta potential of the coated polyplexeswas determined 5 min after their incubation in NaAc buffer containing10% FBS.

Measurement of Cellular Uptake

Cell internalization of III-20% PDL/Cy3-DNA polyplexes was monitoredusing flow cytometry. HEK293 cells were incubated with III-20%PDL/Cy3-DNA polyplexes using the same conditions as the transfectionprocedure. After incubating for 4 h at either 37° C., cells were washedwith PBS and harvested using enzyme-free cell dissociation buffer(Gibco). Cells were washed with 1% BSA in PBS and then analyzed on a BDBiosciences FACScan. To quantify cell association with coated anduncoated polyplexes HEK293 cells were incubated for 4 hours with III-20%PDL/Cy3-DNA polyplexes coated with various polyE-mRGD densities.

Results

Because of its excellent transfection capability and low toxicity,III-20% PDL was tested for in vivo gene therapy by injection into micebearing A549-derived tumor xenografts. First, polyplexes of III-20% PDLterpolymer with luciferase plasmid (pLucDNA) was administered throughthe tail vein: this treatment resulted in limited expression ofluciferase in the tumors (no coat, FIG. 13A). It was possible that lowgene delivery efficiency was caused by 1) the positive charges on thepolyplex surface (the zeta potential of the polyplex: +8.9 mV) (FIG.13B), which attracts and binds with negatively charged plasma proteinsin the blood during circulation, leading to its rapid clearance by thereticuloendothelial system (RES) and 2) the instability of the polyplexnanoparticles. As evidence of instability, polyplex particles incubatedin NaAc buffer solution containing 10% serum nearly doubled in sizewithin 15 minutes and increased by over 10-fold after 75 minutes (FIG.13C). As the result of this increase in size, polyplexes might becleared from the circulation by uptake in the liver.

To improve in vivo gene delivery the surface of III-20% PDL/pLucDNApolyplexes was modified by coating the particles with polyE-mRGD, asynthetic peptide containing three distinct segments. The first segmentis a 16-(amino acid) polyglutamic acid (polyE), which is negativelycharged at physiological pH and, therefore, capable of electrostaticbinding to the positively charged surface of the polyplexes. The secondsegment is a 6-unit neutral polyglycine, which serves as a neutrallinker. The third segment is the amino acid sequencearginine-glycine-aspartic acid-lysine (RGDK, mRGD), which includes theRGD sequence that binds the tumor endothelium through the interaction ofRGD with α_(v)β₃ and α_(v)β₅. In addition, R/KxxR/K allows binding toneuropilin-1 (Sugahara, et al. Tissue-penetrating delivery of compoundsand nanoparticles into tumors. Cancer Cell 16, 510-520 (2009)) (Teesalu,et al. C-end rule peptides mediate neuropilin-1-dependent cell,vascular, and tissue penetration. Proc Natl Acad Sci USA 106,16157-16162 (2009)). Bindings with integrins and neuropilin-1 canimprove tumor-targeted and tissue-penetrating delivery to tumors invivo. Similar approaches have been reported to facilitateligand-specific gene delivery in vitro and targeted gene delivery toliver, spleen, and bone marrow in vivo (Green, et al. Electrostaticligand coatings of nanoparticles enable ligand-specific gene delivery tohuman primary cells. Nano Lett 7, 874-879 (2007)). (Harris, et al.Tissue-specific gene delivery via nanoparticle coating. Biomaterials 31,998-1006 (2010)). Coating with polyE-mRGD reversed the surface charge ofIII-20% PDL/pLucDNA polyplex (FIG. 13B): when polyE-mRGD was added at5:1 peptide/DNA weight ratio, the zeta potential of the polyplex changedfrom +8.9 mV to −5.8 mV.

Peptide coated polyplexes were stable upon incubation in NaAc buffercontaining 10% serum (FIG. 13B) and resistant to aggregation (FIG. 13B),indicating that the modified polyplexes can escape clearance by RESduring circulation in vivo. Resistance to aggregation is particularlyimportant, because small particle size limits clearance by liver andmaintains transfection ability of polyplex particles at the tumor site.It was observed that overcoating of III-20% PDL/pLucDNA polyplexsignificantly decreased transfection (FIG. 13D), despite our observationthat uptake efficiency for overcoated (10×) polyplexes was not reducedin comparison to optimally coated (5×) particles (FIG. 9). On the basisof these results, the ratio of peptide to DNA at 5:1 was selected forthe subsequent in vivo studies.

Compared with the uncoated polyplex particles, intravenousadministration of coated III-20% PDL/pLucDNA polyplexes exhibited˜14,000 times higher gene expression in tumor (FIG. 13A).

Example 4 Terpolymers can Mediate Transfection of Cancer Cells andInhibit Cancer Growth In Vivo

PolyE-mRGD coated polyplex particles of III-20% PDL with therapeutic DNAwere tested for their ability to deliver genes and inhibit tumor growthin vivo. It is well known that tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) can preferentially kill malignanttumor cells, inhibit tumor related angiogenesis, but not harm normalcells (Kagawa, et al. Antitumor activity and bystander effects of thetumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene.Cancer Res 61, 3330-3338 (2001)) (Matsubara, et al. Gene therapy withTRAIL against renal cell carcinoma. Mol Cancer Ther 5, 2165-2171 (2006))(Cantarella, et al. TRAIL inhibits angiogenesis stimulated by VEGFexpression in human glioblastoma cells. Br J Cancer 94, 1428-1435(2006)). In addition to direct tumor cell killing via transfection,TRAIL is also known to induce apoptosis in adjacent tumor cells due to abystander effect (Kagawa, et al. Antitumor activity and bystandereffects of the tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) gene. Cancer Res 61, 3330-3338 (2001)). For this reason, aconstruct containing a fused TRAIL-GFP segment, pEGFP-TRAIL, was chosenas a therapeutic agent. III-20% PDL/pEGFP-TRAIL polyplexes wereprepared, coated with polyE-mRGD, and then injected via tail vein inanimals bearing tumors (Kagawa, et al. Antitumor activity and bystandereffects of the tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) gene. Cancer Res 61, 3330-3338 (2001)).

In preliminary in vitro studies, III-20% PDL complexed with pEGFPdelivered the gene with an efficiency significantly higher than that ofLipofectamine 2000 or PEI (FIG. 14A). Cytotoxicity due to TRAIL wasefficiently achieved in vitro by III-20% PDL/pEGFP-TRAIL polyplexes:three days after transfection, proliferation of A549 cells was inhibitedby 58%, compared with cells treated with a vector control. In contrast,treatments with Lipofectamine/TRAIL and PEI/TRAIL complexes inhibitedcell proliferation by only 21% and 9%, respectively (FIG. 10). Inaddition, 5× coating, which was selected as the optimal for our in vivostudy, did not coat Lipofectamine/DNA complexes and was insufficient tocoat PEI/TRAIL complexes (FIG. 11).

For in vivo tumor treatment, polyE-mRGD coated III-20% PDL/pEGFP-TRAILpolyplexes were administrated three times a week for six weeks at a doseof 1.7 mg per mouse. During the entire course of treatment, notoxicity—as measured by weight loss, for example—was observed (FIG.14B). Treatment of polyE-mRGD coated III-20% PDL/pEGFP-TRAIL polyplexessignificantly inhibited tumor growth. By the end of the experiment, theaverage tumor size in the mouse group treated with the coated TRAILpolyplex was ˜300 mm³, which was significantly smaller than the averagetumor size for the groups treated with control polyplexes (polyE-mRGDcoated III-20% PDL/pLuc) or PBS, which were ˜700 mm³ (p<0.05, one-wayANOVA) (FIG. 14C). Histochemical analysis by TUNEL staining revealed asignificant increase in the number of apoptotic cells after treatmentwith TRAIL (FIG. 14D). The tumor inhibition activity of coated III-20%PDL/pEGFP-TRAIL polyplexes was not due to either naked DNA orpolyE-mRGD, since independently, both of them exhibited limited toxicity(FIG. 12).

Example 5 Synthesis and Purification of Poly(EthyleneGlycol)-Poly(ω-Pentadeca Lactone-Co-N-Methyldiethyleneamine-Co-Sebacate)(PEG-PPMS) Block Copolymers

The block copolymers were synthesized via copolymerization ofω-pentadecalactone (PDL), diethyl sebacate (DES), andN-methyldiethanolamine (MDEA) using MeO-PEG-OH as the chain-terminatingagent and Novozym 435 as the catalyst. The molar ratios of thecomonomers and the PEG substrates are reported in Table 4. The amount ofMeO-PEG-OH with Mn of 5000 Da or 2000 Da (PEG5K or PEG2K) was selectedto form PEG-PPMS block copolymers with approximately 40 wt % PEG uponcomplete conversion of the feeds. The PDL content in the PPMS blocks ofthe copolymers is controlled by adjusting the molar ratio ofPDL/DES/MDEA comonomers. Thus, PDL, DES, MDEA, and PEG5K or PEG2K invarious ratios (Table 4) were blended with Novozym 435 (10 wt % vs.total substrates) and diphenyl ether solvent (200 wt % vs. totalsubstrates) to form reaction mixtures.

All copolymerization reactions were carried out in two stages: firststage oligomerization at 90° C. under 1 atm pressure of nitrogen gas for20 h, and second stage polymerization at 90° C. under 1.4 mmHg vacuumfor 72 h. At the end of the reactions, n-hexane was added to the productmixtures to precipitate the resultant copolymers. The obtained crudeproducts were then washed with fresh n-hexane twice to extract andremove the residual diphenyl ether solvent in the polymers.Subsequently, the copolymers were dissolved in chloroform and werefiltered to remove the catalyst particles. Complete evaporation andremoval of the of the chloroform solvent from the filtrates at 30° C.under high vacuum (<0.5 mmHg) overnight yielded the purified blockcopolymers.

The data on polymer yield, molecular weight, and composition aresummarized in Table 4.

TABLE 1 Characterization of The Purified PEG-PPMS Block CopolymersSynthesized Using Different Feed ratio Feeds (mmol) Polymer Yield PEGContent PGL Content Sample^(a) MeO-PEG-OH PDL DES MDEA (%) M_(w) ^(b)M_(w)/M_(n) ^(c) (wt %) (mol %)^(c) PEG2K-PPMS-11% PDL 0.675 0.73 6.586.24 79 10100 1.5 42 11 PEG2K-PPMS-20% PDL 0.690 1.53 6.12 6.77 80 110001.8 42 20 PEG2K-PPMS-30% PDL 0.708 2.36 1.50 8.14 80 10100 2.0 42 34PEG2K-PPMS-40% PDL 0.715 3.23 4.88 4.52 75 10300 1.8 42 40PEG2K-PPMS-51% PDL 0.725 4.26 4.26 3.90 81 11800 1.8 42 51PEG5K-PPMS-10% PDL 0.270 0.71 6.58 6.43 85 23100 1.6 42 19PEG5K-PPMS-20% PDL 0.276 1.53 6.12 5.98 85 24500 1.5 42 26PEG5K-PPMS-30% PDL 0.282 2.36 5.50 5.36 87 25200 1.5 42 20PEG5K-PPMS-41% PDL 0.286 3.25 1.88 4.72 80 23000 1.5 42 41PEG5K-PPMS-50% PDL 0.280 4.14 4.14 4.00 90 27200 1.6 42 50^(a)PEG2K-PPMS copolymer and PEGSK-PPMS copolymer represent PEG-PPMSblock copolymers consisting of 2000 Da and 5000 Da PEG chain segmentsrespectively. Both PEG2K-PPMS and PEGSK-PPMS copolymers and denoted withx mol % PDL indicating the content of PDL units vs. (PDL + sabacate)units in the PPMS blocks. ^(b)Measured by GPC. ^(c)Mol % PDL units vs.(PDL + sabacate) units in the PPMS chain segments calculated from the ¹HNMR spectra.

PEG-PPMS block copolymer: 1H NMR (CDCl3; ppm) 1.26-1.30 (br.), 1.61 (m,br.), 2.26-2.32 (m), 2.34 (s), 2.70 (t), 3.64 (s), 4.05 (t), 4.16 (t);13C NMR (CDCl3; ppm) 24.84, 24.90, 24.93, 24.99, 25.92, 28.64, 29.04,29.07, 29.13, 29.25, 29.28, 29.47, 29.53, 29.58-29.63 (m), 34.16, 34.21,34.27, 34.32, 42.79, 55.87, 61.86, 64.32, 70.54, 173.55, 173.61, 173.74,173.80.

At the end of the reactions, all formed polymer products prior topurification showed mono-modal molecular weight distributions and noun-reacted MeO-PEG-OH was detected by GPC. During the copolymerization,the amino group of MDEA does not require protection and deprotectionsteps owning to the high tolerance of the enzyme catalyst towards thetertiary amine functionality.

The molecular structures of the PEG-PPMS block copolymers werecharacterized by both 1H and 13C NMR spectroscopy. Consistent with blockcopolymer structures, the copolymer NMR spectra exhibited resonanceabsorptions (e.g., singlet proton resonance at 3.64 ppm and carbon-13resonance at 70.54 ppm) due to poly(ethylene glycol) segments inaddition to the absorbances attributable to randompoly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) chains.No other resonance signals were observed. The PDL contents in PPMS chainblocks of the copolymers were calculated from the copolymer ¹H NMRspectra according to the method reported previously. The PEG contents inthe block copolymers were measured by comparing the intensity of theproton resonance at 3.64 ppm vs. the intensities of the other protonabsorbances due to the repeat units of PPMS blocks. The randomdistribution of PDL, MDEA, and sebacate units in the PPMS chain segmentswas confirmed by ¹³C NMR spectroscopy analysis. Such random structuresare desirable for drug delivery purposes since micelles made of PEG-PPMSblock copolymers with random PPMS chains allow encapsulation and evendistribution of drug molecules in the micelle cores to minimize burstrelease of the drug during delivery.

The PEG-PPMS block copolymers with different compositions were preparedand successfully purified in good yield (up to 90%). During the polymersyntheses, the PEG block length were varied from 2000 Da to 5000 Da.Table 4 summarizes the characterization data on the purified copolymers.The results show that both PEG and PDL contents in the copolymers can bereadily controlled by adjusting the feed ratios employed during thecopolymerization reactions. All synthesized block copolymers containedapproximately 40 wt % PEG (Table 4) since the screening studiesindicated that at this PEG content, PEG-PPMS block copolymers readilyformed stable micelles in aqueous medium (details regarding the micellestability are discussed in a latter section).

Because MeO-PEG-OH can only link to the ends of PPMS chains, thePEG-PPMS copolymers have two possible types of block structures:PEG-PPMS diblock chains and/or PEG-PPMS-PEG triblock chains. ThePEG5K-PPMS and PEG2K-PPMS copolymers at ˜40 wt % PEG content should havemolecular weight values of approximately 12500 Da and 5000 Darespectively if the synthesized copolymers are primarily diblockcopolymers, but would possess molecular weights of approximately 25000Da and 10000 Da correspondingly if the copolymers are primarily triblockcopolymers. The observed molecular weight (M_(w)) values of 23000 to27000 Da for the PEG5K-PPMS copolymers and the values of 10000 to 12000Da for the PEG2K-PPMS copolymers indicate that these block copolymersare substantially rich in PEG-PPMS-PEG triblock chain structures.

Thermal Characterization

The thermal stability of the PEG-PPMS block copolymers was investigatedby thermogravimetric analysis (TGA). The weight loss curves of thecopolymers are depicted in FIG. 15 and the copolymer thermal degradationdata are shown in Table 5. The copolymers are thermally stable up to300° C., start to degrade at about 360° C., and undergo essentiallycomplete thermal degradation at approximately 440° C. The temperature atthe maximal degradation rate (Tmax) is in the range of 408-421° C. forPEG2K-PPMS copolymers and of 413-424° C. for PEG5K-PPMS copolymers. Itis noticeable that for either PEG2K-PPMS or PEG5K-PPMS copolymers, Tmaxgradually increases with increasing PDL content in the polymers (Table5).

TABLE 2 Thermal Properties of The PEG-PPMS Block Copolymers TGAAnalysis^(b) DSC Analysis T_(o) T_(f) T_(max) T_(g) Sample^(a) (° C.) (°C.) (° C.) T_(m) (° C.)^(c) (° C.)^(d) PEG2K-PPMS-11% PDL 366 430 40843.3 PEG2K-PPMS-20% PDL 365 436 413 42.3 PEG2K-PPMS-30% PDL 369 435 41344.0 PEG2K-PPMS-40% PDL 374 439 417 47.5 54.0 PEG2K-PPMS-51% PDL 376 443421 47.2 59.2 PEG5K-PPMS-10% PDL 361 434 413 53.0 PEG5K-PPMS-20% PDL 363436 420 53.2 27.6 PEG5K-PPMS-30% PDL 366 438 420 52.1 33.6PEG5K-PPMS-41% PDL 372 440 423 53.8 50.1 34.4 PEG5K-PPMS-50% PDL 375 442424 54.2 50.4 37.9 ^(a)See Table 1 for the meaning of the abbreviatedsample names. ^(b)T_(o), T_(f), and T_(max) represent the startingdegradation temperature, final degradation temperature, and thetemperature at the maximum degradation rate, respectively. ^(c)T_(m):melting temperature. ^(d)T_(g): glass transition temperature.

As shown in FIG. 15 and Table 5, the effects of PEG chain block lengthon the PEG-PPMS copolymer thermal degradation are minimal. The meltingbehaviors of the copolymers were studied by DSC. The obtained DSC curvesfrom the second heating runs are displayed in FIG. 16, and the meltingtemperatures (T_(m))/glass transition temperatures (T_(g)) of allsamples are summarized in Table 5. The DSC results show that allPEG-PPMS copolymers are semicrystalline materials and several samplespossess more than one melting temperature due to the block structures ofthe polymers. The major melting events at 43-47° C. for PEG2K-PPMScopolymers and those at 53-54° C. for PEG5K-PPMS copolymers areattributable to the PEG crystallites in the copolymers, which aremolecular weight dependent with longer PEG chains increasing the meltingtemperature. The other weaker melting transitions at a high PDL contentare possibly associated with the crystallites formed by PPMS chainsegments in the copolymers. Previous results show thatpoly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate)copolymer with a low (10-20%) PDL content is liquid at 25° C. and doesnot have a melting point above ambient temperature.

Example 6 Preparation of Blank and DTX-Loaded PEG-PPMS Micelles

The micelles were fabricated using a dialysis method. In a typicalexperiment, 38 mg of PEG-PPMS copolymer with or without docetaxel (DTX,2 mg) was dissolved in 1 mL of THF in a sealed vial. The micellizationwas then induced by continuously adding the organic solution into 10 mLof PBS (0.01 M, pH=7.4) using a syringe. The resultant mixture wasstirred at room temperature for 10 min and was subsequently dialyzedwith a dialysis membrane (MWCO 3,500 Da) against 1 L of PBS (0.01 M,pH=7.4) overnight at ambient temperature. To remove the un-entrappeddrug, the micelle solutions were placed in ultrafiltration centrifugetubes (MWCO 100 kDa) and were centrifuged for 30 min at 5000 rpm. Theobtained ultrafiltrates were used for subsequent studies.

For DTX-encapsulated PEG-PPMS micelles, the drug loading and entrapmentefficiency were determined by HPLC (Agilent 1260, USA) equipped with aC18 chromatographic column at 30° C. using 50:50 (v/v)acetonitrile/water mixed solvent as the mobile phase. The mobile phaseflow rate was maintained at 1.0 mL/min. In a typical procedure, 50 μL ofthe above micelle solutions in PBS were diluted in 1 mL of THF and 20 μLof the resultant mixtures were injected to the HPLC during the analyses.The amount of DTX was quantitatively measured by a UV detector at 230 nmwavelength. 30 The drug loading and entrapment efficiency in micellesare calculated according to the following equations:

${{Drug}\mspace{14mu}{{loading}(\%)}} = {\frac{{drug}\mspace{14mu}{amount}\mspace{14mu}{in}\mspace{14mu}{micelles}}{{mass}\mspace{14mu}{of}\mspace{14mu}{micelles}} \times 100\%}$${{Entrapment}\mspace{14mu}{{efficiency}(\%)}} = {\frac{{drug}\mspace{14mu}{amount}\mspace{14mu}{in}\mspace{14mu}{micelles}}{{drug}\mspace{14mu}{feeding}} \times 100\%}$

Characteristics of the DTX-Loaded PEG-PPMS Copolymer Micelles

Docetaxel (a typical anticancer drug) was encapsulated into PEG-PPMScopolymer micelles using a dialysis method. The average size,polydispersity index (PDI), zeta-potential, drug loading, and entrapmentefficiency for all DTX-loaded micelles are summarized in Table 6. Theaverage sizes of the micelles are dependent on the copolymercomposition. 34 The DTX-loaded micelles of all PEG5K-PPMS copolymerswere larger than 300 nm (Table 6).

TABLE 3 Characteristics of The DTX-loaded PEG-PPMS Copolymer MicellesZeta^(b) Drug Entrapment Copolymer^(a) Size (nm) (mV) PDI loading (%)efficiency (%) PEG2K-PPMS-11% PDL 192 ± 3 −8.2 ± 0.6 0.204 2.25 ± 0.0445.8 ± 0.7 PEG2K-PPMS-20% PDL 184 ± 1 −8.5 ± 1.4 0.164 2.31 ± 0.10 47.1± 2.1 PEG2K-PPMS-30% PDL 180 ± 3 −8.7 ± 2.1 0.166 2.34 ± 0.05 47.8 ± 1.0PEG2K-PRMS-40% PDL 174 ± 6  −10 ± 0.6 0.201 2.61 ± 0.10 53.2 ± 2.0PEG2K-PPMS-51% PDL 166 ± 3 −8.5 ± 1.0 0.168 2.92 ± 0.01 60.0 ± 0.2PEG5K-PPMS-10% PDL  404 ± 45 −2.5 ± 0.2 0.592 1.77 ± 0.02 36.1 ± 0.4PEG5K-PPMS-20% PDL  319 ± 10 −1.5 ± 0.1 0.473 1.37 ± 0.06 27.9 ± 1.3PEG5K-PPMS-30% PDL  442 ± 25 −1.7 ± 0.2 0.372 0.94 ± 0.04 19.2 ± 0.8PEG5K-PPMS-41% PDL  343 ± 23 −1.1 ± 0.2 0.172 0.47 ± 0.04  9.5 ± 0.9PEG5K-PPMS-50% PDL  355 ± 17 −1.6 ± 0.2 0.149 0.43 ± 0.02  8.7 ± 0.4^(a)See Table 1 for the meaning of the abbreviated sample names.^(b)Measured in PBS solution (0.01M, pH = 7.4).

In comparison, the average sizes of the PEG2K-PPMS copolymer micelleswere between 160 and 200 nm, which are attributable to the shorterPEG2K-PPMS copolymer chains vs. their counterparts PEG5K-PPMS copolymerchains. The TEM micrograph and size distribution of the DTX-loadedmicelles prepared from PEG2K-PPMS copolymer with 30% PDL content in thePPMS blocks are shown in FIG. 17. Image analysis of the TEM micrographindicated that the micelles possessed 100-150 nm particle sizes with anarrow size distribution. The DTX-loaded micelles were well dispersedand were nearly spherical in shape. There are no significant differencesin morphology among the micelles made from the PEG2K-PPMS copolymerswith different PDL contents.

The surface charges of all micelle samples were slightly negative in PBSsolution (0.01M, pH=7.4) (Table 6), which is beneficial for in vivo drugdelivery applications of the micelles. It is known that nanoparticleswith nearly neutral surface charge (zeta potential between −10 and +10mV) can decrease their uptake by the reticuloendothelial system (RES)and prolong their circulation time in the blood. The negative surfacecharges of the micelles could result from the absorption of HPO₄ ²⁻and/or H₂PO₄ ⁻ anions in PBS by the micelle particles via hydrogenbonding interactions between the anions and the ether groups of PEGshells or the amino groups of PPMS cores. For amphiphilic blockcopolymer micelles, it is anticipated that hydrophilic chain segments(e.g., PEG) in the outer shell of the micelles can shield the charges inthe micelle core with the long chain blocks being more effective inreducing zeta potential than the short chain blocks. Thus, significantlylower zeta potential values were observed for PEG5K-PPMS copolymermicelles as compared to PEG2K-PPMS copolymer micelles (Table 6).

The drug loading and entrapment efficiency for the DTX-loaded micellesof PEG2K-PPMS and PEG5K-PPMS copolymers were determined by HPLC analysis(Table 6). The DTX entrapment efficiency for PEG2K-PPMS copolymermicelles ranges from 46% to 60%, which increases with increasing PDLcontent in the copolymer. This is expected since the PDL-rich blockcopolymers would form more hydrophobic micelle cores, thus attractingmore hydrophobic DTX drug molecules to the micelles. Possibly because ofrelatively low stability of the PEG5K-PPMS copolymer micelles as theresult of their near neutral surface charges, both drug loading andentrapment efficiency appear lower for these micelle samples (Table 6).

The size and zeta potential of the micelles were found to changesignificantly when the pH of the aqueous medium accommodating themicelles was varied. FIG. 18 shows the particle size and zeta potentialof blank PEG2K-PPMS copolymer micelles as a function of the aqueousbuffer pH. The trends in the size-pH and zeta-pH curves are remarkablysimilar for the micelles of the three PEG2K-PPMS copolymers withdifferent PDL contents (11%, 30%, and 51%). It is evident that theaverage size of the micelle samples gradually increases upon decreasingthe medium pH from 7.4 to 5.0, and then remains nearly constant when thepH value is below 5.0 (FIG. 18A). This pH-responsive behavior observedfor the micelles is anticipated since upon decreasing the pH from 7.4 to5.0, the PPMS cores of the micelles become protonated and morehydrophilic, thus absorbing more water molecules from the aqueous mediumto cause swelling of the micelles. It is assumed that the micelle coresare already fully protonated at pH of 5.0, and as the result, the sizesof the micelles remain fairly constant with further decreasing of the ofthe pH from 5.0.

The effects of the PDL content in the PEG2K-PPMS copolymers on themagnitude of the micelle size change between 7.4 and 5.0 pH values arealso notable. With decreasing PDL content and increasing tertiary aminogroup content in the copolymer, the capacity of the micelle cores toabsorb protons and water molecules is expected to increase. Thus, upondecreasing pH from 7.4 to 5.0, the change in average micelle size wasmore significant for PEG2K-PPMS-11% PDL (from 200 nm to 234 nm) ascompared to PEG2K-PPMS-30% PDL (from 184 nm to 214 nm) andPEG2K-PPMS-51% PDL (from 163 nm to 182 nm) (FIG. 18A).

The zeta potential of the micelles in aqueous medium also exhibitssubstantial pH-dependence (FIG. 18B). At physiological and alkaline pH(7.4 to 8.5), the surface charges of blank PEG2K-PPMS copolymer micelleswere negative, which changed to positive when the pH of the mediumdecreased to acidic range (4.0-6.0). For example, the micelles ofPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL possessedzeta potential values of −5.8, −7.1, −5.1 mV, respectively, at pH of7.4, which turned to +7.6, +5.8, +4.0 mV, correspondingly, at a lower pHof 5.0. On the basis of the above discussions, this surface chargedependence on pH is attributable to the protonation or deprotonation ofthe PPMS cores of the micelles at different medium pH. At an alkaline pH(7.4-8.5), most of the amino groups in the micelles presumably are notprotonated, and the micelle particles remain negatively charged due tothe absorption of HPO⁴²⁻ and/or H₂PO⁴⁻ anions in PBS by the micelles. Inparticular, at pH of 8.5, the zeta-potential values were −8.1 mV, −7.9mV, −9.0 mV for PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, andPEG2K-PPMS-51% PDL, respectively (FIG. 18B). Upon decreasing pH from 7.4to 5.0, the tertiary amino moieties in the micelle PPMS cores becomemostly protonated, turning the micelles to positively charged particles.

Consistently, among the three micelle samples, PEG2K-PPMS-11% PDLmicelles with the largest capacity to absorb protons displayed thehighest zeta potential values at pH of 4.0-5.0, whereas PEG2K-PPMS-51%PDL micelles with the smallest protonation capacity showed the lowestzeta potentials (FIG. 18B). The observed micelle surface chargeresponses to the medium pH are highly desirable since the negativesurface charge of the micelles at physiological pH can alleviate theinteraction of the micelles with serum protein in the blood and prolongtheir in vivo circulation time. On the other hand, the reverse topositive surface charge at the tumor extracellular pH of approximately6.5 could enhance the uptake of these micelles by target tumor cells.

Example 7 Measurement of Critical Micelle Concentration (CMC) Values ofPEG-PPMS Block Copolymers

The pH-responsive behavior and CMC values of the copolymer micelles werestudied using fluorospectrophotometry. Pyrene was employed as thefluorescence probe. In a typical experiment, an aliquot of pyrenesolution in THF was added to tube containers. After the THF solventevaporated completely, aqueous micelle solutions with various polymerconcentrations in predetermined amounts were added to each tuberespectively and were vortexed to yield a final pyrene concentration of6.0×10-7 M. The solutions were kept at room temperature for 24 h toensure that pyrene dissolution in aqueous media reaches the equilibriumbefore measurement. Fluorescence intensities of the pyrene solubilizedin the micelle cores were determined by a fluorescence steady-statesystem with excitation at 334 nm wavelength and emission detection inthe range from 350 to 420 nm wavelength. It is known that only at aconcentration above the CMC, micelles with a hydrophobic core and ahydrophilic shell are formed. The entrapment of pyrene molecules in thecores of the micelles leads to a higher intensity of the third peak (I3)at 391 nm in the emission spectrum. Therefore, the CMC values can beevaluated by monitoring the intensity of the third peak, which isusually normalized by the intensity of the first emission peak (I₁) at371 nm. In actual practice, for a given PEG-PPMS micelle sample, theI3/I1 intensity ratios are plotted against the logarithm of polymerconcentrations, and the CMC value was determined from the intersectionof the best-fit lines, which corresponds to the minimum polymerconcentration required for formation of stable micelles in the aqueousmedium.

The self-assembling ability and pH-responsive micellization behaviors ofPEG2K-PPMS copolymers were investigated in PBS solutions (0.01 M) withpH of 7.4 or 5.0 using fluorospectrophotometry. The fluorescenceintensity ratio (I3/I1) of dissolved pyrene as a function of thelogarithm of the copolymer concentration is shown in FIG. 19 for thecopolymer samples PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, andPEG2K-PPMS-51% PDL. The cross points of the curves correspond to the CMCvalues of the polymer samples, above which stable PEG2K-PPMS copolymermicelles are formed and the concentration of entrapped pyrene in themicelles increases with increasing copolymer concentration. The CMCvalues of PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDLwere respectively 5.50, 2.04, and 1.95 μg/mL at pH of 7.4, which changedcorrespondingly to 18.2, 14.1, and 10.7 μg/mL at pH of 5.0. This resultcan be explained as follows. A higher PDL content in the PPMS chainsegments of the PEG2K-PPMS copolymer enhances the hydrophobicity of themicelle inner cores, thus increasing the micelle stability (ordecreasing the CMC) in aqueous medium. On the other hand, for a givenPEG2K-PPMS copolymer, the stability of the copolymer micelles can alsosubstantially affected by the medium pH. As discussed in the previoussection, upon decreasing the medium pH from 7.4 to 5.0, the PPMS coresof the copolymer micelles become protonated and less hydrophobic whichwould, of course, reduce the stability of the micelles. CMC is animportant parameter indicative of micelle stability, and micelles with alow CMC is often required for effective in vivo drug deliveryapplications since such micelles would not disintegrate upon dilution inthe blood stream after intravenous injection. The results of this studyindicate that the PEG2K-PPMS copolymer micelles are sufficiently stablecarriers for in vivo drug delivery applications.

Example 8 Evaluation of In Vitro Micelle Stability

Micelle solutions at 1 mg/mL concentration were prepared by mixingdifferent PEG-PPMS copolymer micelles with a pre-determined amount ofPBS solution (0.01 M, pH=7.4) containing 10 vol % human serum or FBS.The micelle solutions were gently stirred at 37° C. for 144 h. Atvarious time intervals, aliquots were withdrawn from the solutions andwere analyzed by DLS to measure the average particle size of thePEG-PPMS copolymer micelles. All measurements were performed intriplicate.

In order for micelles to be suitable as carriers to deliver drugs invivo, the micelle particles must possess sufficient stability inserum-containing aqueous medium. To evaluate the stability of PEG2K-PPMScopolymer micelles under realistic conditions for in vivo drug deliveryapplications, the blank micelles were incubated with PBS solution(0.01M, pH=7.4) containing either 10 vol % human serum or 10 vol % FBSand the average size of each micelle sample was monitored subsequentlyfor 6 days. As shown in FIG. 20, the micelle sizes of all threePEG2K-PPMS copolymer samples (PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL,and PEG2K-PPMS-51% PDL) remained fairly constant during the whole periodof the incubation time. Thus, these PEGylated poly(amine-co-ester)terpolymer micelles can stay intact with minimal interactions with serumproteins in the media, which is critically important for achievingprolonged circulation time of the micelles in the blood to effectivelydeliver drugs to target tissue/cells.

Example 9 In Vitro DTX Release from the Drug-Loaded PEG-PPMS Micelles

In vitro DTX release studies from the micelles were performed bydialyzing the DTX-encapsulated micelles against PBS (0.01 M) solutionwith a pH of 5.0 or 7.4.32 Typically, 400 μL of each micelle sample at40 mg/mL polymer concentration was placed in a dialysis bag (MWCO 3,500Da) which was then incubated in 10 ml of PBS solution (containing 0.5%Tween 80) at 37° C. and 100 rpm shaking speed. At different timeintervals, aliquot samples of 1.0 mL volume were withdrawn from the PBSand the same amount of the fresh buffer was added after each samplewithdrawal. Sink condition was maintained throughout the drug releaseperiod. The aliquot samples were centrifuged at 12,000 rpm for 5 min andthe supernatant was analyzed in triplicate by HPLC to quantify theamount of released DTX as described above.

As discussed above, the physical properties (e.g., size and zetapotential) of the copolymer micelles are dependent on the pH of themedium accommodating the micelles. Therefore, variations in the rate ofDTX release from PEG2K-PPMS copolymer micelles at different pH areanticipated. In vitro drug release behaviors of the DTX-encapsulatedmicelles of PEG2K-PPMS copolymer samples (PEG2K-PPMS-11% PDL,PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL) were studied in PBS solutionat both physiological pH of 7.4 and acidic pH of 5.0.

FIG. 21 depicts the accumulative values (in percentage) of DTX releasedfrom the micelles during a period of 144 h. In general, the DTX releasefrom all micelle samples followed biphasic release kinetics and indeedexhibited remarkable pH-dependence. The DTX-loaded PEG2K-PPMS copolymermicelles release 25-45% drug rapidly during the initial 12 h, followedby a more gradual release of additional 25-40% drug for the subsequent132 h (FIG. 21). The influence of the medium pH on the drug release rateis substantial. For example, at the end of the incubation period (144h), the values of accumulated DTX released from the micelles ofPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDLcopolymers are respectively 66%, 60%, and 55% at physiological pH of7.4, which increase correspondingly to 85%, 81%, and 75% at acidic pH of5.0 (FIG. 21). The observed pH-triggered acceleration of DTX releasefrom the PEG2K-PPMS copolymer micelles is consistent with the earlierobservation that changing of the medium pH from 7.4 to 5.0 causessignificant swelling of the micelles due to the protonation and sizeincrease of the micelle PPMS cores. This pH-triggered micelle sizeexpansion would certainly facilitate the diffusion and release ofentrapped DTX from the micelle cores to the aqueous medium. On the otherhand, at a given pH, the DTX release rate is presumably controlled bythe interactions between the drug and the PPMS matrix in the micellecores. Since PDL-rich PEG2K-PPMS copolymers are expected to form stronghydrophobic domains in their micelle inner cores to better trap andretain hydrophobic DTX molecules, the drug release from such copolymermicelles should be more gradual and sustained. This hypothesis issupported by the experimental result showing that at both pH of 7.4 and5.0, the DTX release rate from PEG2K-PPMS copolymer micelles decreaseswith increasing PDL content in the PPMS chain segments of the copolymer(FIG. 21).

It is known that upon uptake of micelles by tumor cells, the micelleparticles are subjected to entrapment in endosomes with pH ranging from5.5 to 6.0 and in lysosomes with pH ranging from 4.5 to 5.0. As theabove results clearly show, these acidic environments would inevitablytrigger fast DTX release from PEG2K-PPMS copolymer micelles, thusenhancing the cytotoxicity of the drug-loaded micelles. The amino groupsin the copolymers would act as proton sponges to facilitate endosomalescape. Therefore, the pH-responsive properties exhibited by thePEG2K-PPMS copolymer micelles are highly desirable, which render them tobe superior carriers for delivery of anticancer drugs.

In Vitro Cellular Uptake Studies

For cellular uptake studies, fluorescent dye coumarin-6 (C6) with lowwater-solubility was used as a probe to imitate DTX and was encapsulatedinto PEG-PPMS copolymer micelles using the same protocol for preparationof the DTX-loaded micelles as described in an earlier section. Thephysicochemical properties of the C6-loaded micelles were characterized.SK-BR-3 cells were seeded into 24-well plates at 2.0×105 cells/well (0.5mL) and allowed to attach overnight. Free C6 or C6 entrapped in themicelles was then added to each well at 0.2 μg/mL C6 concentration andthe cells were incubated at 37° C. for 1 h, 4 h, and 8 h. A same amountof C6 was used to treat each group of the cells. For free C6, a DMSOsolution of 1 mg/mL was prepared and was diluted with DMEM to therequired concentration. After the incubation, C6-containing media wereremoved and the cells were thoroughly rinsed twice with cold PBSsolution. Subsequently, the treated cells were harvested using trypsinand centrifuged at 1500 rpm for 5 min. Upon removal of the supernatants,the cells were resuspended in 0.3 ml of PBS solution and were analyzedin groups containing 10,000 cells by FACScan (Becton Dickinson, SanJose, Calif.) to measure cellular uptake efficiency of the PEG-PPMScopolymer micelles. Intracellular C6 was excited with an argon laser of488 nm wavelength at constant intensity and the fluorescence emissionwas detected at 585 nm wavelength.

Confocal laser scanning microscopy (CLSM) was used to visualize theinternalization of C6-loaded micelles by SK-BR-3 cells. Cells wereseeded on glass-bottom dishes containing culture medium and wereincubated overnight at 37° C. C6-loaded micelles were added to each dishat a C6 concentration of 0.2 μg/mL and the cells were incubated foranother 4 h at 37° C. Subsequently, the medium was removed and the cellswere washed twice with PBS solution. After staining the cell nuclei withHochest 33342 (10 μg/mL) and the acidic late endosomes and lysosomeswith LysoTracker Red (75 nM), the intracellular distribution of C6 wasobserved by CLSM using a Lecia TCS SP5 Spectral Confocal Microscope witha 63× objective and a diode laser. The excitation wavelength fordetecting C6, Hochest 33342, and LysoTracker Red was 467 nm, 405 nm, and577 nm, respectively.

The cellular uptake efficiency of the drug-loaded PEG2K-PPMS copolymermicelles was investigated with both flow cytometry and confocal laserscanning microscopy (CLSM). To simplify the experimental processes,coumarin-6 (C6, a hydrophobic fluorescent marker) was employed insteadof DTX and was encapsulated in PEG2K-PPMS copolymer micelles using thesame method for preparing the DTX-loaded micelles as described in theExperimental Section. Because of its substantially low solubility inwater, C6 is often considered as a non-releasing agent in nanoparticlesamples, which is idea for study on cellular uptake of the micelleparticles. To ensure that the C6-encapsulated PEG2K-PPMS copolymermicelles are comparable to their DTX-loaded micelle counterparts, thephysicochemical properties of C6-loaded micelles were characterized. Theresults showed that for a given PEG2K-PPMS copolymer, the average size,PDI, and zeta-potential were very similar between the C6-loaded micellesand the DTX-loaded micelles. For example, the mean sizes of theC6-loaded micelles were respectively 194±4 nm, 171±5 nm, and 210±8 nm,and the C6 entrapment efficiency of the micelles were correspondingly81% (±1.3%), 96% (±2.0%) and 99% (±1.5%) for the copolymersPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL. Theaccumulative values of C6 release from all three micelle samples wereless than 1% over a period of 10 h incubation time.

Flow cytometry analysis was performed to investigate cellular uptakeefficiency of the C6-loaded PEG2K-PPMS copolymer micelles at differenttime intervals. FIG. 22 shows the mean fluorescence intensity (MFI)values of SK-BR-3 cells incubated with free C6 or C6-loaded micelles at37° C. for up to 8 h. It is evident that the cellular uptake process istime-dependent with an increasing number of the micelles being absorbedby the cells at an extended time. Among the three micelle samples, theuptake rate of the micelles by SK-BR-3 cells (measured by MFI of thecells) follows the order: PEG2K-PPMS-30% PDL copolymer micelles<PEG2K-PPMS-51% PDL copolymer micelles <PEG2K-PPMS-11% PDL copolymermicelles (FIG. 22). At a given incubation time, the MFI value for thecell group treated with PEG2K-PPMS-11% PDL copolymer micelles isstatistically higher than that of the cells treated with free C6,PEG2K-PPMS-30% PDL copolymer micelles, or PEG2K-PPMS-51% PDL copolymermicelles. This result was confirmed by the CLSM images of SK-BR-3 cellsafter absorbing C6-loaded PEG2K-PPMS copolymer micelles. When treatedwith each of the three PEG2K-PPMS micelle samples containing same amountof C6, the cell group incubated with C6-encapsulated PEG2K-PPMS-11% PDLcopolymer micelles displayed strongest green fluorescence intensity. Thecellular uptake experiments also indicate that the PEG2K-PPMS copolymermicelles can be quickly absorbed by the cells since after 1 h shortincubation time, the cell groups treated with each of the three micellesamples exhibited substantially higher MFI values than the cells treatedwith free C6 (FIG. 22).

To clarify whether or not drug-loaded PEG2K-PPMS copolymer micelles canescape from endosomes and lysosomes after cellular uptake, the locationsof C6-loaded micelles in SK-BR-3 cells relative to the positions ofacidic endosomes and lysosomes were visualized by staining the endosomesand lysosomes with LysoTracker Red and were observed with CLSM. TheC6-loaded PEG2K-PPMS copolymer micelles were internalized by the cellsand distributed all over the cytoplasm with only few of them found toco-localize (thus possibly still trapped/associated) with theLysoTracker Red-stained organelles after 4 h incubation. This resultshows that the PEG2K-PPMS copolymer micelles are capable of escapingfrom the endosomes and lysosomes likely due to the good buffercapability of the tertiary amine groups in the copolymers which act asproton sponges.

The cellular uptake efficiency of the PEG2K-PPMS copolymer micellescould be affected by several factors, including the particle size,particle surface properties (e.g., surface charge and hydrophobicity),and polymer composition. As discussed earlier, the average particlesizes of the three C6-loaded micelle samples are around 200 nm andcomparable. Thus, the observed variations in cellular uptake efficiencyare possibly attributed to the differences in the micelles' surfaceproperties and their polymer composition.

Example 10 In Vitro Cytotoxicity Study

The in vitro cytotoxicity of free DTX, blank PEG-PPMS copolymer micellesand DTX-loaded copolymer micelles were tested against SK-BR-3 cellsusing the standard MTT assay. To evaluate the pH-responsivecharacteristics of the DTX-loaded micelles, the cytotoxicity experimentswere performed at different pH values as follows. The cells were seededin a 96-well plate at a density of 2,500 cells per well in 100 μL ofRPMI-1640 medium with 10% FBS, and were kept at 37° C. in 5% CO₂atmosphere. After allowing the cells to adhere overnight, 100 μL of themedium containing different amount of DTX (either free drug or inPEG-PPMS copolymer micelles) was added to each well. The medium pH wasadjusted with 0.1 M HCl or 0.1 M NaOH to a desired value (7.4 or 6.5)right before the treatment. When blank PEG-PPMS copolymer micelles wereused instead of the DTX-loaded micelles, the concentration of bothmicelles was kept same. After 48 h of incubation, the medium was removedand the cells were rinsed twice with PBS solution. Subsequently, 100 μLof the fresh medium and 20 μL of PBS containing 5 mg/mL MTT were addedto each well, and the plate was incubated for additional 4 h. Afterremoval of the medium in each well, 150 μL of DMSO was added to dissolvethe formazan salt crystals and the absorbance of each well at 570 nmwavelength was measured using a microplate reader (Bio Tek Synergy4).All assays were carried out in triplicate and were repeated at leasttwice. Cell viability was calculated as the value in percentage of(absorbance of the cells treated)/(absorbance of the cells culturedwithout treatment).

The cytotoxicity of blank PEG2K-PPMS copolymer micelles, DTX-loadedPEG2K-PPMS copolymer micelles, and free DTX was evaluated on SK-BR-3cells at pH of 7.4 using the MTT assay, and the results are shown inFIG. 23. The blank micelles exhibited no obvious cytotoxicity on SK-BR-3cells as the cell viabilities of all treated cell groups were over 90%(FIG. 23A). For example, after treatment with the micelles ofPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDLcopolymers at concentration as high as 200 μg/mL, the three treated cellgroups had respectively 97%, 94%, and 90% of the cells remaining viable.FIG. 23B depicts the cytotoxicity of the DTX-loaded micelles and freeDTX incubated with SK-BR-3 cells for 48 h. As expected, the cellviability decreases with increasing concentration of DTX either in theform of the free drug or in the form of the drug encapsulated in themicelles. To quantify the in vitro efficacy of these micelleformulations, IC50 values for DTX-loaded micelles of copolymersPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS −51% PDL werecalculated to be 0.57 nM, 0.83 nM, and 1.42 nM, respectively. Incomparison, the IC50 value for free DTX was found to be 1.27 nM. IC50 isdefined as the drug concentration of a specific formulation which isrequired to kill 50% of cells after they are incubated with the drug fora designated time period (48 h). Thus, except the PEG2K-PPMS-51% PDLcopolymer micelles, both DTX-loaded PEG2K-PPMS-11% PDL copolymermicelles and DTX-loaded PEG2K-PPMS-30% PDL copolymer micelles possesssignificantly higher cytotoxicity against SK-BR-3 cells than free DTX.The exceptionally high efficacy observed for DTX-loaded PEG2K-PPMS-11%PDL copolymer micelles is likely attributed to fast cellular uptake ofthe micelles (FIG. 22) and anticipated rapid intracellular DTX releasefrom the micelles upon entrapment of the micelle particles in acidicendosomes/lysosomes (FIG. 21). The rate of DTX release from themicelles, particularly pH-triggered acceleration of the drug release,appears to play a more important role than the cellular uptake ininfluencing the cytotoxicity of the DTX-loaded micelles. Thus, althoughthe cellular uptake is faster for DTX-loaded PEG2K-PPMS-51% PDLcopolymer micelles vs. DTX-loaded PEG2K-PPMS −30% PDL copolymer micelles(FIG. 22), the latter micelles release the drug at a higher rate toexert higher cytotoxicity toward SK-BR-3 cells (FIG. 21).

To elucidate how the cell viability is affected by the release andcellular uptake of the drug in an acidic tumor extracellular matrixenvironment, the cytotoxicity of the DTX-loaded PEG2K-PPMS copolymermicelles toward SK-BR-3 cells were also investigated at pH of 6.5. Theobtained IC50 values at pH of 6.5 for free DTX and DTX-loaded micellesof copolymers PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51%PDL were correspondingly 1.34, 0.45, 0.58, and 1.20 nM. Thus, all threemicelle samples exhibited decreased IC50 values and enhancedcytotoxicity upon changing of the medium pH from 7.4 to 6.5. Thisacid-promoted, cytotoxicity enhancement for the PEG2K-PPMS copolymermicelles appears to be independent on the concentration of theencapsulated drug. For example, at various doses (0.05-10 nM) of DTXencapsulated in PEG2K-PPMS-11% PDL copolymer micelles, the viability ofthe SK-BR-3 cells treated at acidic pH of 6.5 was always lower than thatof the cells treated at pH of 7.4 (FIG. 23C). This acid-enhancedcytotoxicity presumably results from increased cellular uptakeefficiency of the micelles upon changing the medium pH from slightlybasic (pH of 7.4) to mildly acidic (pH of 5.0-6.5). As shown in FIG.18B, when the medium pH was adjusted from 7.4 to 6.5, the surfacecharges of the PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, andPEG2K-PPMS-51% PDL copolymer micelles changed from negative values tonear neutral or positive charges, which should promote binding of themicelles with the cell membrane and accelerate the cellular uptake ofthe micelles at the lower pH.

Example 11 Erythrocyte Agglutination and Hemolysis

The erythrocyte agglutination and hemolytic activity of PEG-PPMScopolymer micelles were investigated using literature procedures. Humanblood was centrifuged at 1000 rpm for 5 min to isolate erythrocytes(RBC). The obtained erythrocytes were washed in isotonic PBS (0.01 M,pH=7.4) until the supernatant was clear. The cells were then diluted inPBS to decrease the cell density to 3×10⁷ cells/mL. This erythrocytesolution was always freshly prepared and was used within 24 h aftercollection. Blank PEG-PPMS copolymer micelle solutions with variouspolymer concentrations were added to the erythrocytes and the resultantmixtures were incubated at 37° C. for 2 h. Triton X-100 (0.1%, w/v) andisotonic PBS were used as positive control and negative control,respectively. At the end of the incubation period, the cell/polymermixed solutions were transferred to a 24-well plate and were observedwith an optical microscope. Meanwhile, the absorbance at 413 nm wasmeasured for the supernatant of each sample using a microplate reader.

The relative hemolytic activity (%) is calculated using the followingequation:

${{Hemolysis}(\%)} = {\frac{A_{Sample} - A_{PBS}}{A_{Triton} - A_{PBS}} \times 100\%}$where A_(Sample), A_(PBS), A_(Triton) represent the absorbance intensityvalues of the supernatants from RBC treated with PEG-PPMS copolymermicelles, PBS, and Triton X-100, respectively.

Hemolysis and agglutination behaviors are two major biocompatibilityissues that frequently occur during in vivo application of new drugformulations. To verify that the PEG2K-PPMS copolymer micelles arebiocompatible, the blank micelles at various concentrations wereincubated with erythrocytes to evaluate the tendency of the copolymersto induce hemolysis and agglutination. Erythrocytes treated withPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL copolymermicelles, along with PBS solution, showed no agglutination and theerythrocytes retained the integrity of double-concave disc shape. Thequantitative hemolysis results on the copolymer micelles are illustratedin FIG. 24. Although the hemolysis activity (percentage ratio vs. TritonX-100 activity) of the three micelle samples increased slightly withincreasing micelle concentration, the hemolysis ratios forPEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL copolymermicelles were respectively 4.5%, 4.6%, and 3.2% even at the highestpolymer concentration of 200 μg/ml. These hemolysis values aresubstantially lower than the maximum nonhemolytic value (8%) set by ASTMF756-08 standard test method. According to the method, samples with ahemolysis ratio below 8% are considered nonhemolytic. Thus, the PEG-PPMScopolymers can be defined as nonhemolytic with minimal tendency to causeagglutination.

A series of PEGylated poly(amine-co-ester) terpolymers were successfullysynthesized in one step via lipase-catalyzed copolymerization of PDL,DES, and MDEA comonomers in presence of poly(ethylene glycol) methylether as a chain-terminating agent. The resultant amphiphilic PEG-PPMSblock copolymers consisted of hydrophilic PEG chain segments withmolecular weight (Mn) ranging from 2000 Da to 5000 Da and hydrophobicrandom PPMS copolymer chain segments with PDL content ranging from 10 to50 mol %. TGA analysis shows that the block copolymers are thermallystable up to 300° C. with the fastest degradation taking place at408-424° C. Consistent with the copolymer block structures, a majormelting temperature at 43-54° C. due to PEG crystallites was observedfor all PEG-PPMS copolymers, and an additional weak melting event wasdetected for the PDL-rich copolymers (e.g., PEG2K-PPMS-40% PDL,PEG2K-PPMS-51% PDL, PEG5K-PPMS −41% PDL, and PEG5K-PPMS-50% PDL)possibly due to the presence of second crystallites formed by PDL-richPPMS chain segments.

Upon dissolution in water, the PEG2K-PPMS copolymers with 42 wt % PEGcontent readily formed micelles with average particle size in the rangebetween 160 and 190 nm, which were stable at physiological pH of 7.4 inpresence of serum proteins. The PEG2K-PPMS copolymers possessed highblood compatibility and exhibited minimal activity to induce hemolysisand agglutination. The copolymer micelles are pH-responsive: withdecreasing the medium pH from 7.4 to 5.0, the sizes of the micellesincreased significantly while the micelle surface charges reversed fromnegative charges to positive charges. Correspondingly, DTX-encapsulatedPEG2K-PPMS copolymer micelles showed gradual sustained drug release atphysiological pH of 7.4, but remarkably accelerated DTX release atacidic pH of 5.0. Cellular uptake study revealed that the drug-loadedmicelle particles were readily absorbed by SK-BR-3 cells. CLSMexperiments indicated that the micelles were able to escape fromentrapment by endosomes and lysosomes after the cellular uptake. Becauseof these desirable properties, DTX-loaded micelles of PEG2K-PPMScopolymers (e.g., PEG2K-PPMS-11% PDL and PEG2K-PPMS-30% PDL) with lowPDL content, high protonation capability, and fast drug release at anacidic pH exert substantially higher potency against SK-BR-3 cancercells than free DTX drug. These results demonstrate that PEG2K-PPMScopolymer micelles have great potential to serve as pH-responsivenano-carriers for controlled release delivery of DTX to treat cancers.

We claim:
 1. A method of making polymers, the method comprising mixingan enzyme catalyst with monomeric units under conditions forming apolymer of Formula I,

wherein the polymer degrades by hydrolysis, wherein x, y, and q areindependently integers from 1-1000, wherein Z is O or NR′, wherein R′ ishydrogen, substituted or unsubstituted alkyl, or substituted orunsubstituted aryl, wherein the monomer units are selected from thegroup consisting of one or more lactones having the formula

one or more diacids or diesters, having the formula

one or more an amine-diols, triamines, or hydroxy diamines, having theformula

wherein R is (CH₂)_(n); wherein R^(x) is hydrogen or alkyl; wherein R″substituted or unsubstituted alkyl, or substituted or unsubstitutedaryl; wherein each X is independently OH or N(R′″)₂, wherein R′″ ishydrogen, substituted or unsubstituted alkyl, or substituted orunsubstituted aryl; wherein n is an integer from 1-30; each occurrenceof m, o, and p are independently integers from 1-20; w is an integerfrom 1 to
 1000. 2. The method of claim 1, wherein the enzyme catalyst isa lipase.
 3. The method of claim 2, wherein the lipase is lipase B fromCandida antarctica.
 4. The method of claim 1, wherein the molar ratiosof the lactone/diacid or diester/amine diol or triamine or hydroxydiamine is from about 10:90:90 to about 90:10:10.
 5. The method of claim1, wherein the weight average molecular weight of the polymers, asmeasured by gel permeation chromatography using narrow polydispersitypolystyrene standards, is from about 10,000 Daltons to about 50,000Daltons.
 6. The method of claim 1, comprising (i) incubating the monomerunits and enzyme catalyst at a temperature from between about 80° C. andabout 90° C., at about 1 atm N₂.
 7. The method of claim 6, furthercomprising, after step (i), (ii) incubating the monomer units and enzymecatalyst at a temperature from between about 80° C. and about 90° C., atabout 1.6 mm Hg.
 8. The method of claim 3, wherein n is 4, 10, 13, or14.
 9. The method of claim 8, wherein m is 5, 6, or
 7. 10. The method ofclaim 9, wherein R″ is substituted or unsubstituted alkyl.
 11. Themethod of claim 10, wherein the alkyl is methyl, ethyl, propyl, n-butyl,or t-butyl.
 12. The method of claim 8, wherein R″ is substituted orunsubstituted aryl.
 13. The method of claim 12, wherein the aryl isphenyl.
 14. The method of claim 4, wherein n is 4, 10, 13, or
 14. 15.The method of claim 14, wherein m is 5, 6, or
 7. 16. The method of claim15, wherein R″ is substituted or unsubstituted alkyl.
 17. The method ofclaim 16, wherein the alkyl is methyl, ethyl, propyl, n-butyl, ort-butyl.
 18. The method of claim 15, wherein R″ is substituted orunsubstituted aryl.
 19. The method of claim 18, wherein the aryl isphenyl.
 20. The method of claim 1, wherein the polymer has a molecularweight between 6,800 and 19,000 Da.
 21. The method of claim 20, whereinR^(x) is hydrogen.
 22. The method of claim 21, wherein m is 7, n is 13,o and p are 2, and R″ is substituted or unsubstituted alkyl.